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Metabolism-dependent taxis and control of motility in Pseudomonas putida
Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
2013 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Bacteria living in soil and aquatic habitats rapidly adapt to changes in physico-chemical parameters that influence their energy status and thus their ability to proliferate and survive. One immediate survival strategy is to relocate to more metabolically optimal environments. To aid their movement through gradients (a process called taxis), many bacteria use whip like flagella organelles. Soil-dwelling Pseudomonas putida possesses a polar bundle of flagella that propel the bacterium forward in directed swimming motility. P. putida strains are generally fast growing, have a broad metabolic capacity, and are resistant to many harmful substances – qualities that make them interesting for an array of industrial and biotechnological application. This thesis identifies some of the factors that are involved in controlling the flagella driven motility of P. putida.

In the first part of the thesis, I present evidence that P. putida displays energy-taxis towards metabolisable substrates and that the surface located Aer2 receptor (named after its similarities to the Escherichia coli Aer receptor) is responsible for detecting the changes in energy-status and oxygen-gradients that underlie this response. Aer2 is expressed simultaneously with the flagella needed for taxis responses and its expression is ensured during nutrient scares conditions through the global transcriptional regulators ppGpp and DksA.

In addition to Aer2, P. putida possesses two more Aer-like receptors (Aer1 and Aer3) that are differentially expressed. Like Aer2, Aer1 and Aer3 co-localize to one cell pole. Although the signals to which Aer1 and Aer3 respond are unknown, analysis of Aer1 uncovered a role in motility control for a protein encoded within the same operon. This protein, called PP2258, instigated the work described in the second part of my thesis on the involvement of the second messenger c-di-GMP in regulation of P. putida motility. Genetic dissection of the catalytic activities of PP2258 revealed that it has the unusual capacity to both synthesize and degrade c-di-GMP. Coupling of the c-di-GMP signal originating from PP2258 to motility control was traced to the c-di-GMP binding properties of the protein PP4397. In the last part of the thesis, I present possible mechanisms for how these different components might interact to create a signal transduction cascade – from the surface located Aer1 receptor to PP2258 and the c-di-GMP responsive PP4397, and from there to the flagella motors – to ultimately determine flagella performance and the motility status of P. putida.

Place, publisher, year, edition, pages
Umeå: Umeå universitet , 2013. , 48 p.
Keyword [en]
Motility, c-di-GMP, metabolism-dependent taxis, receptors, transcription, Pseudomonas putida
National Category
Microbiology
Research subject
Molecular Biology
Identifiers
URN: urn:nbn:se:umu:diva-66138ISBN: 978-91-7459-563-5 (print)OAI: oai:DiVA.org:umu-66138DiVA: diva2:605802
Public defence
2013-03-21, Norrlands universitetssjukhus, Byggnad 6L, Major Groove, Umeå universitet, Umeå, 09:00 (English)
Opponent
Supervisors
Available from: 2013-02-28 Created: 2013-02-15 Last updated: 2013-02-20Bibliographically approved
List of papers
1. Metabolism-dependent taxis towards (methyl)phenols is coupled through the most abundant of three polar localized Aer-like proteins of Pseudomonas putida
Open this publication in new window or tab >>Metabolism-dependent taxis towards (methyl)phenols is coupled through the most abundant of three polar localized Aer-like proteins of Pseudomonas putida
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2008 (English)In: Environmental Microbiology, ISSN 1462-2912, E-ISSN 1462-2920, Vol. 10, no 5, 1320-1334 p.Article in journal (Refereed) Published
Abstract [en]

Comparatively little is known about directed motility of environmental bacteria to common aromatic pollutants. Here, by expressing different parts of a (methyl)phenol-degradative pathway and the use of specific mutants, we show that taxis of Pseudomonas putida towards (methyl)phenols is dictated by its ability to catabolize the aromatic compound. Thus, in contrast to previously described chemoreceptor-mediated chemotaxis mechanisms towards benzoate, naphthalene and toluene, taxis in response to (methyl)phenols is mediated by metabolism-dependent behaviour. Here we show that P. putida differentially expresses three Aer-like receptors that are all polar-localized through interactions with CheA, and that inactivation of the most abundant Aer2 protein significantly decreases taxis towards phenolics. In addition, the participation of a sensory signal transduction protein composed of a PAS, a GGDEF and an EAL domain in motility towards these compounds is demonstrated. The results are discussed in the context of the versatility of metabolism-dependent coupling and the necessity for P. putida to integrate diverse metabolic signals from its native heterogeneous soil and water environments.

Place, publisher, year, edition, pages
John Wiley & Sons, 2008
National Category
Microbiology
Identifiers
urn:nbn:se:umu:diva-20467 (URN)10.1111/j.1462-2920.2007.01546.x (DOI)18279347 (PubMedID)
Available from: 2009-03-20 Created: 2009-03-20 Last updated: 2017-12-13Bibliographically approved
2. The σ-factor FliA, ppGpp and DksA coordinate transcriptional control of the aer2 gene of Pseudomonas putida
Open this publication in new window or tab >>The σ-factor FliA, ppGpp and DksA coordinate transcriptional control of the aer2 gene of Pseudomonas putida
2010 (English)In: Environmental Microbiology, ISSN 1462-2912, E-ISSN 1462-2920, Vol. 12, no 6, 1439-1451 p.Article in journal (Refereed) Published
Abstract [en]

Here the σ-factor requirement for transcription of three similar, but differentially regulated, aer genes of Pseudomonas putida KT2440 is investigated. Previous work has shown that the three Aer proteins, like chemoreceptors, colocalize to a single pole in a CheA-dependent manner. Lack of Aer2 - the most abundant of these three proteins - mediates defects in metabolism-dependent taxis and aerotaxis, while lack of Aer1 or Aer3 has no apparent phenotype. We show, using wild-type and mutant P. putida derivatives combined with P. putida reconstituted FliA- (σ28) and σ70-dependent in vitro transcription assays, that transcription of aer2 is coupled to motility through the flagella σ-factor FliA, while σ70 is responsible for transcription of aer1 and aer3. By comparing activities of the wild-type and mutant forms of the aer2 promoter, we present evidence (i) that transcription from FliA-dependent Paer2 is enhanced by changes towards the Escherichia coli consensus for FliA promoters rather than towards that of P. putida, (ii) that the nature of the AT-rich upstream region is important for both output and σ70 discrimination of this promoter, and (iii) that Paer2 output is directly stimulated by the bacterial alarmone ppGpp and its cofactor DksA.

Place, publisher, year, edition, pages
John Wiley & Sons, 2010
National Category
Microbiology
Identifiers
urn:nbn:se:umu:diva-33333 (URN)10.1111/j.1462-2920.2009.02139.x (DOI)000278394400007 ()20089044 (PubMedID)
Note

E-pub ahead of print

Available from: 2010-04-21 Created: 2010-04-21 Last updated: 2017-12-12Bibliographically approved
3. Genetic dissection of a motility-associated c-di-GMP signalling protein of Pseudomonas putida
Open this publication in new window or tab >>Genetic dissection of a motility-associated c-di-GMP signalling protein of Pseudomonas putida
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2013 (English)In: Environmental Microbiology Reports, ISSN 1758-2229, E-ISSN 1758-2229, Vol. 5, no 4, 556-565 p.Article in journal (Refereed) Published
Abstract [en]

Lack of the Pseudomonas putidaPP2258 protein or its overexpression results in defective motility on solid media. The PP2258 protein is tripartite, possessing a PAS domain linked to two domains associated with turnover of c-di-GMP - a cyclic nucleotide that controls the switch between motile and sessile lifestyles. The second messenger c-di-GMP is produced by diguanylate cyclases and degraded by phosphodiesterases containing GGDEF and EAL or HD-GYP domains respectively. It is common for enzymes involved in c-di-GMP signalling to contain two domains with potentially opposing c-di-GMP turnover activities; however, usually one is degenerate and has been adopted to serve regulatory functions. Only a few proteins have previously been found to have dual enzymatic activities - being capable of both synthesizing and hydrolysing c-di-GMP. Here, using truncated and mutant derivatives of PP2258, we show that despite a lack of complete consensus in either the GGDEF or EAL motifs, the two c-di-GMP turnover domains can function independently of each other, and that the diguanylate cyclase activity is regulated by an inhibitory I-site within its GGDEF domain. Thus, motility-associated PP2258 can be added to the short list of bifunctional c-di-GMP signalling proteins.

National Category
Microbiology
Identifiers
urn:nbn:se:umu:diva-66137 (URN)10.1111/1758-2229.12045 (DOI)
Funder
Swedish Research Council, 621-2008-3557Swedish Research Council, 2011-4791
Available from: 2013-02-15 Created: 2013-02-15 Last updated: 2017-12-06Bibliographically approved
4. PP4397 provides the link between PP2258 c-di-GMP signalling and altered motility in Pseudomonas putida
Open this publication in new window or tab >>PP4397 provides the link between PP2258 c-di-GMP signalling and altered motility in Pseudomonas putida
(English)Manuscript (preprint) (Other academic)
National Category
Microbiology
Identifiers
urn:nbn:se:umu:diva-66136 (URN)
Funder
Swedish Research Council, 2011-4791
Available from: 2013-02-15 Created: 2013-02-15 Last updated: 2013-02-20Bibliographically approved

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