Second messenger control of mRNA translation by dynamic ribosome modification

Abstract

8Control of mRNA translation is a crucial regulatory mechanism used by bacteria to respond to their 9 environment. In the soil bacterium Pseudomonas fluorescens, RimK modifies the C-terminus of ribosomal 10 protein RpsF to influence important aspects of rhizosphere colonisation through proteome remodelling. In 11 this study, we show that RimK activity is itself under complex, multifactorial control by the co-transcribed 12 phosphodiesterase trigger enzyme (RimA) and a polyglutamate-specific protease (RimB). Furthermore, 13 biochemical experimentation and mathematical modelling reveal a role for the nucleotide second messenger 14 cyclic-di-GMP in coordinating these activities. Active ribosome regulation by RimK occurs by two main routes: 15 indirectly, through changes in the abundance of the global translational regulator Hfq and directly, with 16 translation of surface attachment factors, amino acid transporters and key secreted molecules linked 17 specifically to RpsF modification. Our findings show that post-translational ribosomal modification functions 18 as a rapid-response mechanism that tunes global gene translation in response to environmental signals. 19 20 42 forms a highly complex, non-linear and pleiotropic network, with multiple connections to other signalling 43 systems and phenotypic outputs that vary profoundly in response to environmental cues (25, 26). The model 44 P. fluorescens strain SBW25, for example, contains over 40 cdG-metabolic enzymes (27) that influence 45 phenotypes at every regulatory level and whose expression varies throughout rhizosphere colonisation (22). 46Pseudomonas cdG signalling shows extensive overlap with other global gene regulators, such as Gac/Rsm 47 (28, 29) and the RNA-chaperone Hfq (30). 48The small hexameric protein Hfq facilitates binding between mRNA and regulatory sRNAs (31, 32) and 49 controls biofilm formation (33), carbon catabolite repression (34), amino-acid transport (35, 36), virulence 50 (37) and motility (36). In P. fluorescens, Hfq is important for niche adaptation, with deletion mutants 51 displaying strongly reduced motility, increased surface attachment, and severely compromised rhizosphere 52 colonisation (25, 30). The regulatory connections between cdG and Hfq are reflected in the close phenotypic 53 parallels between mutants in both pathways (30, 38, 39). 54We recently identified a further contributor to the post-transcriptional regulatory network in Pseudomonas 55 spp. (30). Similar to Hfq and cdG, the ribosomal modification protein RimK controls the transition between 56 active and sessile bacterial lifestyles, with an SBW25 rimK mutant affected in motility, root attachment and 57 amino-acid uptake, and showing reduced rhizosphere colonisation efficiency. Deletion of rimK in pathogenic 58Pseudomonas spp. leads to significantly reduced virulence and cytotoxicity (30). RimK is an ATP-dependent 59 glutamyl ligase that adds glutamate residues to the C-terminus of ribosomal protein RpsF, which in-turn 60 induces specific changes in the bacterial pro...