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Global regulatory factors that impact metabolic and lifestyle choices in Pseudomonas putida
Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
2017 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

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.

Place, publisher, year, edition, pages
Umeå: Umeå Universitet , 2017. , p. 62
Keywords [en]
Pseudomonas putida, c-di-GMP, motility, ppGpp, DksA, biofilm dispersal, transcriptional and translational regulation, dmp-system, phenol catabolism, carbon repression control, plasmid stability
National Category
Biological Sciences Microbiology
Identifiers
URN: urn:nbn:se:umu:diva-141912ISBN: 978-91-7601-801-9 (print)OAI: oai:DiVA.org:umu-141912DiVA, id: diva2:1157257
Public defence
2017-12-08, A103 (Astrid Fagraeus), byggnad 6A, Norrlands Universitetssjukhus, Umeå, 09:00 (English)
Opponent
Supervisors
Available from: 2017-11-17 Created: 2017-11-15 Last updated: 2018-06-09Bibliographically approved
List of papers
1. PP4397/FlgZ provides the link between PP2258 c-di-GMP signalling and altered motility in Pseudomonas putida
Open this publication in new window or tab >>PP4397/FlgZ provides the link between PP2258 c-di-GMP signalling and altered motility in Pseudomonas putida
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2018 (English)In: Scientific Reports, ISSN 2045-2322, E-ISSN 2045-2322, Vol. 8, article id 12205Article in journal (Refereed) Published
Abstract [en]

Bacteria swim and swarm using rotating fagella that are driven by a membrane-spanning motor complex. Performance of the fagella motility apparatus is modulated by the chemosensory signal transduction system to allow navigation through physico-chemical gradients – a process that can be fne-tuned by the bacterial second messenger c-di-GMP. We have previously analysed the Pseudomonas putida signalling protein PP2258 that has the capacity to both synthesize and degrade c-di-GMP. A PP2258 null mutant displays reduced motility, implicating the c-di-GMP signal originating from this protein in control of P. putida motility. In Escherichia coli and Salmonella, the PilZ-domain protein YcgR mediates c-di-GMP responsive control of motility through interaction with the fagellar motors. Here we provide genetic evidence that the P. putida protein PP4397 (also known as FlgZ), despite low sequence homology and a diferent genomic context to YcgR, functions as a c-di-GMP responsive link between the signal arising from PP2258 and alterations in swimming and swarming motility in P. putida.

Place, publisher, year, edition, pages
Nature Publishing Group, 2018
National Category
Biological Sciences
Identifiers
urn:nbn:se:umu:diva-141908 (URN)10.1038/s41598-018-29785-w (DOI)000441625500028 ()
Funder
Swedish Research Council, 2011-4791/2016-02047The Kempe Foundations
Note

Originally included in thesis in manuscript form

Available from: 2017-11-15 Created: 2017-11-15 Last updated: 2018-09-05Bibliographically approved
2. The stringent response promotes biofilm dispersal in Pseudomonas putida
Open this publication in new window or tab >>The stringent response promotes biofilm dispersal in Pseudomonas putida
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2017 (English)In: Scientific Reports, ISSN 2045-2322, E-ISSN 2045-2322, Vol. 7, article id 18055Article in journal (Refereed) Published
Abstract [en]

Biofilm dispersal is a genetically programmed response enabling bacterial cells to exit the biofilm in response to particular physiological or environmental conditions. In Pseudomonas putida biofilms, nutrient starvation triggers c-di-GMP hydrolysis by phosphodiesterase BifA, releasing inhibition of protease LapG by the c-di-GMP effector protein LapD, and resulting in proteolysis of the adhesin LapA and the subsequent release of biofilm cells. Here we demonstrate that the stringent response, a ubiquitous bacterial stress response, is accountable for relaying the nutrient stress signal to the biofilm dispersal machinery. Mutants lacking elements of the stringent response – (p)ppGpp sythetases [RelA and SpoT] and/or DksA – were defective in biofilm dispersal. Ectopic (p)ppGpp synthesis restored biofilm dispersal in a ∆relA ∆spoT mutant. In vivo gene expression analysis showed that (p)ppGpp positively regulates transcription of bifA, and negatively regulates transcription of lapA and the lapBC, and lapE operons, encoding a LapA-specific secretion system. Further in vivo and in vitro characterization revealed that the PbifA promoter is dependent on the flagellar σ factor FliA, and positively regulated by ppGpp and DksA. Our results indicate that the stringent response stimulates biofilm dispersal under nutrient limitation by coordinately promoting LapA proteolysis and preventing de novo LapA synthesis and secretion.

Place, publisher, year, edition, pages
Nature Publishing Group, 2017
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:umu:diva-141910 (URN)10.1038/s41598-017-18518-0 (DOI)000418644100014 ()29273811 (PubMedID)
Note

Originally published in thesis in manuscript form.

Available from: 2017-11-15 Created: 2017-11-15 Last updated: 2018-06-09Bibliographically approved
3. Multiple Hfq-Crc target sites are required to impose catabolite repression on (methyl)phenol metabolism in Pseudomonas putida CF600
Open this publication in new window or tab >>Multiple Hfq-Crc target sites are required to impose catabolite repression on (methyl)phenol metabolism in Pseudomonas putida CF600
2018 (English)In: Environmental Microbiology, ISSN 1462-2912, E-ISSN 1462-2920, Vol. 20, no 1, p. 186-199Article in journal (Refereed) Published
Abstract [en]

The dmp-system encoded on the IncP-2 pVI150 plasmid of Pseudomonas putida CF600 confers the ability to assimilate (methyl)phenols. Regulation of the dmp-genes is subject to sophisticated control, which includes global regulatory input to subvert expression of the pathway in the presence of preferred carbon sources. Previously we have shown that in P. putida, translational inhibition exerted by the carbon repression control protein Crc operates hand-in-hand with the RNA chaperon protein Hfq to reduce translation of the DmpR regulator of the Dmp-pathway. Here we show that Crc and Hfq co-target four additional sites to form riboprotein complexes within the proximity of the translational initiation sites of genes encoding the first two steps of the Dmp-pathway to mediate two-layered control in the face of selection of preferred substrates. Furthermore, we present evidence that Crc plays a hitherto unsuspected role in maintaining the pVI150 plasmid within a bacterial population, which has implications for (methyl)phenol degradation and a wide variety of other physiological processes encoded by the IncP-2 group of Pseudomonas-specific mega-plasmids.

Keywords
catabolite repression, translational regulation, Crc, CrcZ, CrcY, Hfq, Phenol catabolism, IncP-2 plasmids
National Category
Cell and Molecular Biology
Identifiers
urn:nbn:se:umu:diva-141902 (URN)10.1111/1462-2920.13966 (DOI)000419784100015 ()29076626 (PubMedID)
Available from: 2017-11-15 Created: 2017-11-15 Last updated: 2018-06-09Bibliographically approved

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