Adaptive Remodeling of the Bacterial Proteome by Specific Ribosomal Modification Regulates Pseudomonas Infection and Niche Colonisation

Abstract

Post-transcriptional control of protein abundance is a highly important, underexplored regulatory process by which organisms respond to their environments. Here we describe an important and previously unidentified regulatory pathway involving the ribosomal modification protein RimK, its regulator proteins RimA and RimB, and the widespread bacterial second messenger cyclic-di-GMP (cdG). Disruption of rimK affects motility and surface attachment in pathogenic and commensal Pseudomonas species, with rimK deletion significantly compromising rhizosphere colonisation by the commensal soil bacterium P. fluorescens, and plant infection by the pathogens P. syringae and P. aeruginosa. RimK functions as an ATP-dependent glutamyl ligase, adding glutamate residues to the C-terminus of ribosomal protein RpsF and inducing specific effects on both ribosome protein complement and function. Deletion of rimK in P. fluorescens leads to markedly reduced levels of multiple ribosomal proteins, and also of the key translational regulator Hfq. In turn, reduced Hfq levels induce specific downstream proteomic changes, with significant increases in multiple ABC transporters, stress response proteins and non-ribosomal peptide synthetases seen for both ΔrimK and Δhfq mutants. The activity of RimK is itself controlled by interactions with RimA, RimB and cdG. We propose that control of RimK activity represents a novel regulatory mechanism that dynamically influences interactions between bacteria and their hosts; translating environmental pressures into dynamic ribosomal changes, and consequently to an adaptive remodeling of the bacterial proteome.

Fig 1. RimABK is important for P. fluorescens rhizosphere colonisation.

1A. The SBW25 rimABK operon consists of three co-transcribed genes. Gene numbers and predicted translated proteins are shown in each case. 1B. Wheat root attachment by the ΔrimABK and complementation strains relative to SBW25 WT. 1C. Swarming motility of the ΔrimABK and complementation strains relative to SBW25 WT. 1D. Rhizosphere colonisation competition assays. The graph shows the ratio of SBW25 WT or ΔrimABK to WT-lacZ colony forming units (CFU) recovered from the rhizospheres of wheat plants seven days post-inoculation. Each dot represents CFU recovered from an individual plant. Statistically significant differences between SBW25 and ΔrimABK strains are indicated (*** = p < 0.01, * = p < 0.05) in each case. 1E. SBW25 rimK mRNA abundance determined by qRT-PCR, for wheat rhizospheres sampled at various intervals post-inoculation. Expression of rimK is shown relative to M9 0.4% pyruvate liquid-culture (LC).

Fig 2. RimK is important for P. syringae and P. aeruginosa plant infection.

2A. Swarming motility of Pto DC3000 and PA01 ΔrimK relative to their respective WT strains. 2B. Congo Red binding of Pto DC3000 and PA01 ΔrimK compared with their respective WT strains. 2C. Representative spray-infected Arabidopsis Col-0 plants 4 days post-infection with Pto DC3000 WT/ΔrimK. Disease symptoms are less marked with ΔrimK infection. 2D. log (P. syringae CFU per cm² leaf tissue) recovered from Arabidopsis Col-0 plants infected with Pto DC3000 WT or ΔrimK, 2 and 3 days post-infection (dpi). The infection method in each case is stated beneath the graph. 2E. Lettuce leaf infections with P. aeruginosa WT/ΔrimK strains. Lesions photographed after 5 days. 2F. β-hemolysis by P. aeruginosa WT/ΔrimK strains after 24 h growth on horse blood agar.

Fig 3. Biochemical analysis of RimK.

3A. ATPase activity of RimK_Pf incubated with RpsF_Pf and glutamate. RimK_Pf specific activity (nmol ATP hydrolyzed/min/mg RimK_Pf) is shown for increasing concentrations of ATP (open circles). Addition of RpsF_Pf (triangles), glutamate (filled circles) or both (square) increases the V_max of RimK_Pf ATPase activity. 3B. Glutamation assays with E. coli and SBW25 RimK and RpsF. The contents of each assay are indicated underneath the relevant lanes. Independent preparations of RimK_Pf and RpsF_Pf were used in the two panels, which were run separately and are shown side by side for comparative purposes only. Running positions of RimK, RpsF and glutamated-RpsF (RpsF**) are marked with arrows. 3C. Glutamation assays with RimK_Pf and RpsF_Pf. The contents of each assay are indicated, with 0.2, 2.0 and 20 mM glutamate added to the test samples as shown. Running positions of RimK, RpsF and glutamated-RpsF (RpsF*) are marked. 3D. Glutamation assays with RimK_Pf, RpsF_Pf and U-¹⁴C- glutamate. The contents of each reaction is indicated underneath the relevant lane. Control samples were incubated overnight, while time-course samples show 5, 10, 30, 60, 180 minutes, and overnight incubation. The left hand panel shows an overlay of Coomassie stained and radiolabel visualizations of a single gel. The right hand panel shows radiolabel incorporation into RpsF alone.

Fig 4. RimA, RimB and cdG impacts on RimK activity.

4A. ATPase activity of RimK_Pf incubated with RimB, BSA and glutamate. RimK_Pf specific activity (nmol ATP hydrolyzed/min/mg RimK_Pf) is shown for increasing concentrations of ATP (red, circles). Addition of RimB (purple, up-triangles) increases the RimK_Pf V_max, while BSA (brown, down-triangles) does not. RimB alone displays no ATPase activity (green, squares). 4B. ATPase activity of RimK_Pf incubated with RimA and glutamate. RimK_Pf specific activity (nmol ATP hydrolyzed/min/mg RimK_Pf) is shown for increasing concentrations of ATP (purple, squares). Addition of RimA (green, up-triangles) increases the RimK_Pf V_max, while RimA alone displays no ATPase activity (yellow, down-triangles). 4C. Thin layer chromatography of α-³²P-labeled cdG incubated with BSA, YhjH or RimA for the time periods shown. The product of cdG hydrolysis; pGpG, migrates further than cdG but less than α-³²P-GTP. 4D. Biotinylated-cdG pulldown for RimK_Pf. E. coli overexpression cell lysate (RimK sample) is loaded alongside the washed cdG-bead sample (b-cdG pulldown). RimK and streptavidin are indicated with arrows. 4E. SPR sensorgram and affinity data for RimK_Pf binding to biotinylated cdG. A range of RimK_Pf concentrations was used (0.156, 0.312, 0.625, 1.25, 2.5, 5, and 10 μM) and concentration replicates included as appropriate together with buffer only controls. Protein binding and dissociation phases are shown. For the affinity fit, binding responses were measured 4s before the end of the injection and K_d values for each protein calculated using BiaEvaluation software and confirmed by GraphPad. 4F. The effect of cdG addition on glutamation of RpsF_Pf by RimK_Pf. The contents of each reaction is indicated underneath the relevant lanes. Control samples were incubated overnight, while time-course samples show 5, 10, 30, 60, 180 minutes, and overnight incubation. The panel shows an overlay of Coomassie staining and radiolabel visualization (red) of the same gel, as with Fig 3C.

Fig 5. The RimK regulon in Pseudomonas fluorescens.

5A. Up and down-regulated proteins in the ΔrimK mutant compared with SBW25 WT. Pie chart sections indicate the proportion of significantly up- or down-regulated proteins in each functional category as shown. The numbers in each section of the chart refer to the total number of proteins in that category. Interesting or important functional groups are expanded from the chart in each case. 5B. RT-PCR showing relative mRNA abundance in SBW25 WT (+) or ΔrimK (-) for selected RimK-regulated proteins. The housekeeping gene rpoD (σ⁷⁰) and rimK are included as controls. Down-regulated proteins in ΔrimK are shown in red, up-regulated proteins in green. (PFLU)-0068 and 6091 encode ABC transporter components, 3222 an NRPS subunit.

Fig 6. Comparison of the P. fluorescens ΔrimK, Δhfq and rpsF-D139K mutant strains.

6A. Swarming motility of Δhfq relative to SBW25 WT. 6B. Congo Red binding of Δhfq, compared to SBW25 WT and ΔrimK. 6C. Glutamation assays with E. coli and SBW25 RimK, and RpsF/RpsF-D139K. The contents of each assay are indicated underneath the relevant lanes. Running positions of RimK, RpsF and glutamated-RpsF (RpsF*) are marked with arrows. Incubation time is shown above the gel image; all controls were incubated overnight. 6D. Wheat root attachment assay for ΔrimK, Tn7::rimK and rpsF-D139K, relative to WT SBW25. 6E. Rhizosphere colonisation competition assays for Δhfq, ΔrimK, rpsF-D139K and WT SBW25. The graph shows the ratio of mutant to WT-lacZ CFU recovered from the rhizospheres of wheat plants seven days post-inoculation. Each dot represents CFU recovered from an individual plant. 6F. Western blot showing RpsF levels in mutant cell lysates. Statistically significant differences between WT SBW25 and mutant strains are indicated throughout (*** = p < 0.01).

Fig 7. A model for RimABK function in Pseudomonas plant interactions.

During early stage colonisation/initial infection (top row), increased RimK activity leads to increased RpsF glutamation. This leads to increased Hfq levels, with the resulting translational repression promoting phenotypes important for niche colonisation and the establishment of infection, including motility and virulence [4,44,51]. In the established rhizosphere/plant infection environment (bottom row), rimK transcription decreases. Less RpsF is glutamated, leading to altered ribosomal protein abundance and ribosome function, and lower Hfq levels. Release of Hfq translational repression leads to increased production of amino-acid transporters (ABC), oxidative stress response pathways (SodA), NRPSs and attachment factors. These changes promote resource acquisition, stress resistance and root attachment, and prioritize long-term adaptation to the plant environment. RimK activity is further regulated by direct interaction with the RimA/B proteins and the signalling molecule cdG. ‘+/-‘ denotes cases where the nature of protein/dinucleotide control is currently undefined.