Global regulatory factors that impact metabolic and lifestyle choices in Pseudomonas putida

Sammanfattning: Pseudomonas putida strains have a broad metabolic capacity and are innately resistant to many harmful substances – properties that make them of interest for a number of industrial and biotechnological application. They can rapidly adapt to changes in physico-chemical parameters in the soil and water environments they naturally inhabit. Like other bacteria, they have evolved both specific and cross-acting global regulatory circuits to control endurance traits and life style choices in order to survive. Three such survival tactics are 1) the ability to control flagella-mediated motility to search for metabolically favourable locations, 2) to produce protective biofilm structures to resist environmental insults, and 3) to distinguish the energetically most favourable carbon source amongst an array on offer. These processes are often co-ordinated regulated by intersecting networks that are controlled by global signalling molecules (second messengers) such as the nucleotides ppGpp and c-di-GMP, and globally acting proteins.In the first part of my thesis I present evidence that the PP4397 protein of P. putida is responsible for slowing down flagella-driven motility in response to c-di-GMP signalling from a dual-functional c-di-GMP turnover protein termed PP2258. This connection is expanded upon to present a potential signal transduction pathway from a surface located receptor to PP2258 and the c-di-GMP responsive PP4397 protein, and from there to the flagella motors to determine flagella performance. The transcriptional regulatory studies that accompany this work suggest a means by which transcriptional control may serve to initiate a co-ordinated blocking of de novo flagella biogenesis and slowing-down flagella rotation – two processes needed to enter the biofilm mode of growth. Exiting from a biofilm matrix is also a c-di-GMP elicited behaviour, prompted when nutrients become scarce. In my second piece of work I present evidence that hunger-signals in the form of ppGpp directly control transcription to elevate the levels of a c-di-GMP degrading protein – BifA – which lies at the heart of programed biofilm dispersal. The final part of my thesis, concerns how the global regulatory proteins Hfq and Crc act at multiple levels to subvert catabolism of phenolics to favour other preferred sources of carbon. Evidence is presented that this involves a two-tiered translational repression – one at the level of the master regulator of the system, and another at the level of the catabolic enzymes. This study also revealed a hitherto unsuspected role of Crc in maintenance of an IncP-2 plasmid within a bacterial population. This latter finding has implications for a wide variety of processes encoded by the IncP-2 group of Pseudomonas-specific mega-plasmids.