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The versatile role of quorum sensing and phenazines in the biocontrol strain Pseudomonas sp. CMR12a

Katrien De Maeyer UGent (2012)
abstract
Pseudomonas sp. CMR12a was isolated from cocoyam roots in Cameroon, and has excellent biocontrol properties against the cocoyam root rot disease caused by Pythium myriotylum. CMR12a excretes phenazines, redox-active compounds with antibiotic activities; and cyclic lipopeptides, amphiphillic compounds with surface active properties. At first place, this thesis shows the involvement of both phenazines and the cyclic lipopeptide ‘sessilin’ in biocontrol of Rhizoctonia root rot on bean, by using mutants in both compounds, namely CMR12a-ΔPhz and CMR12a-CLP1. Moreover, sessilin has a direct antagonistic effect on the mycelium of R. solani. The main goal of this work, however, was to cross the borders of the yet well-known antagonistic interactions in biological control, and to study the integration of the biocontrol strain in the complex network of soil-plant-bacterium. Therefore, a study of the regulatory systems in CMR12a was indispensable. Two quorum sensing systems were identified in CMR12a, which coordinate expression of their regulon in response to cell density via signal molecules. The PhzI/R system controls phenazine production as in many other Pseudomonas biocontrol strains, while the novel CmrI/R system is assumed to control the balance between the biofilm-promoting sessilin and the swarming-promoting motilin. Interestingly, the latter system controls sessilin/motilin production in accordance with the nutrient status. More specifically, it is assumed that by coordinating a change in the glutamine/glutamate ratio, a change in the sessilin/motilin ratio is realized. In addition, the cognate signal molecule of the CmrI/R system, namely 3-OH-C12-HSL, benefits from motilin production for its diffusion, and promotes plant growth and root length elongation of lettuce and Chinese cabbage seedlings. Besides the nutrient status, this study also shows that the oxygen status has major implications for Pseudomonas sp. CMR12a. In the wet tropical soil, CMR12a probably faces regular periods of limited oxygen availability. The ability to respire nitrate in a process called ‘denitrification’ is a great adaptation of CMR12a to these conditions, which has not often been described for other Pseudomonas biocontrol strains. Interestingly, the cocoyam rhizosphere harbors many antagonistic, phenazine producing, denitrifying Pseudomonas strains. Methylamine and methanol are breakdown products of plant detritus in tropical soils, which are very rich in organic matter, with methanol also being a typical compound occurring at oxic-anoxic interfaces. The CMR12a genome sequence reveals genes involved in oxidation of these compounds, however, their functionality could not be unambiguously proved. metabolic activity was observed on methylamine, while methanol stimulates cell aggregation and biofilm formation. It is assumed that methanol oxidation rather contributes to survival than to growth. Alternatively, CMR12a might use formaldehyde, the reaction product of methanol and methylamine oxidation, which could be performed by other methylotrophic microorganisms in situ. Production of phenazines is considered as another adaptation to microaerobic conditions. Besides their well-known antibiotic effect and involvement in rhizosphere competence, this work addresses the functional role of these compounds for CMR12a’s cell physiology, which might additionally provide another underlying mechanism of the rhizosphere competence of phenazine producing strains. More specifically, the phenazines produced by CMR12a, phenazine-1-carboxylate (PCA) and phenazine-1-carboxamide (PCN), contribute to redox homeostasis under microaerobic conditions. In practical terms this means that the phenazines get intracellularly reduced, and subsequently transfer the electrons to oxygen after diffusion to more oxygen-rich sites. They oxidize NADH when the terminal electron acceptor oxygen becomes limiting, and prevent as such an increase of the NADH/NAD+ ratio and a reduction of the cytoplasm. Probably, a reduced cytoplasm counteracts a remarkable trait of CMR12a, namely the tendency to form spontaneous GacS/GacA mutations. The Gac system is a major regulator of secondary metabolism, including phenazine production, which has been described before to be susceptible to spontaneous mutations resulting in phenotypic switches called phase variation. It was shown that a gac mutant has a severely reduced cytoplasm, and that the stability of the gac locus in CMR12a possibly depends on the reduction status of the cytoplasm. Therefore, different frequencies of Gac-mutated CMR12a cultures were observed under aerobic and microaerobic conditions. Moreover, since phenazines are involved in maintaining the appropriate redox conditions of the cytoplasm, different frequencies of Gac-mutated cultures of CMR12a mutants with altered phenazine production (absence and overproduction of phenazines) were observed. The overproduction of phenazines in mutants in cytochrome o ubiquinol oxidase (CMR12a-PCyo) and in phosphoenolpyruvate phospho-transferase (CMR12a-PtsP) is thought to be a reaction of the cell against the reduction of the cytoplasm caused by the mutation. Possible regulatory mechanisms involved in this fine-tuning of phenazine production are considered. This work has provided more insight into the tropical biocontrol strain Pseudomonas sp. CMR12a in many aspects. The genome revealed special gene clusters and a novel quorum sensing system, which might ultimately be the result of special adaptations to the environment. The physiological relevance of phenazine reduction in Pseudomonas sp.CMR12a has been explored and may be contributing to rhizosphere competence of phenazine producing strains in general.
Please use this url to cite or link to this publication:
author
promoter
UGent
organization
alternative title
De veelzijdige rol van quorum sensing en fenazines in de biologische bestrijdingsstam Pseudomonas sp. CMR12a
year
type
dissertation (monograph)
subject
keyword
biological control, phenazines, pseudomonas species, quorum sensing
pages
III, 217 pages
publisher
Ghent University. Faculty of Bioscience Engineering
place of publication
Ghent, Belgium
defense location
Gent : Faculteit Bio-ingenieurswetenschappen (A0.030)
defense date
2012-01-19 16:00
ISBN
9789059894938
language
English
UGent publication?
yes
classification
D1
additional info
dissertation consists of copyrighted material
copyright statement
I have transferred the copyright for this publication to the publisher
id
1989132
handle
http://hdl.handle.net/1854/LU-1989132
date created
2012-01-17 10:52:59
date last changed
2012-01-19 09:22:25
@phdthesis{1989132,
  abstract     = {Pseudomonas sp. CMR12a was isolated from cocoyam roots in Cameroon, and has excellent biocontrol properties against the cocoyam root rot disease caused by Pythium myriotylum. CMR12a excretes phenazines, redox-active compounds with antibiotic activities; and cyclic lipopeptides, amphiphillic compounds with surface active properties. At first place, this thesis shows the involvement of both phenazines and the cyclic lipopeptide {\textquoteleft}sessilin{\textquoteright} in biocontrol of Rhizoctonia root rot on bean, by using mutants in both compounds, namely CMR12a-\ensuremath{\Delta}Phz and CMR12a-CLP1. Moreover, sessilin has a direct antagonistic effect on the mycelium of R. solani. The main goal of this work, however, was to cross the borders of the yet well-known antagonistic interactions in biological control, and to study the integration of the biocontrol strain in the complex network of soil-plant-bacterium. Therefore, a study of the regulatory systems in CMR12a was indispensable. Two quorum sensing systems were identified in CMR12a, which coordinate expression of their regulon in response to cell density via signal molecules. The PhzI/R system controls phenazine production as in many other Pseudomonas biocontrol strains, while the novel CmrI/R system is assumed to control the balance between the biofilm-promoting sessilin and the swarming-promoting motilin. Interestingly, the latter system controls sessilin/motilin production in accordance with the nutrient status. More specifically, it is assumed that by coordinating a change in the glutamine/glutamate ratio, a change in the sessilin/motilin ratio is realized. In addition, the cognate signal molecule of the CmrI/R system, namely 3-OH-C12-HSL, benefits from motilin production for its diffusion, and promotes plant growth and root length elongation of lettuce and Chinese cabbage seedlings. 
Besides the nutrient status, this study also shows that the oxygen status has major implications for Pseudomonas sp. CMR12a. In the wet tropical soil, CMR12a probably faces regular periods of limited oxygen availability. The ability to respire nitrate in a process called {\textquoteleft}denitrification{\textquoteright} is a great adaptation of CMR12a to these conditions, which has not often been described for other Pseudomonas biocontrol strains. Interestingly, the cocoyam rhizosphere harbors many antagonistic, phenazine producing, denitrifying Pseudomonas strains. Methylamine and methanol are breakdown products of plant detritus in tropical soils, which are very rich in organic matter, with methanol also being a typical compound occurring at oxic-anoxic interfaces. The CMR12a genome sequence reveals genes involved in oxidation of these compounds, however, their functionality could not be unambiguously proved. metabolic activity was observed on methylamine, while methanol stimulates cell aggregation and biofilm formation. It is assumed that methanol oxidation rather contributes to survival than to growth. Alternatively, CMR12a might use formaldehyde, the reaction product of methanol and methylamine oxidation, which could be performed by other methylotrophic microorganisms in situ. 
Production of phenazines is considered as another adaptation to microaerobic conditions. Besides their well-known antibiotic effect and involvement in rhizosphere competence, this work addresses the functional role of these compounds for CMR12a{\textquoteright}s cell physiology, which might additionally provide another underlying mechanism of the rhizosphere competence of phenazine producing strains. More specifically, the phenazines produced by CMR12a, phenazine-1-carboxylate (PCA) and phenazine-1-carboxamide (PCN), contribute to redox homeostasis under microaerobic conditions. In practical terms this means that the phenazines get intracellularly reduced, and subsequently transfer the electrons to oxygen after diffusion to more oxygen-rich sites. They oxidize NADH when the terminal electron acceptor oxygen becomes limiting, and prevent as such an increase of the NADH/NAD+ ratio and a reduction of the cytoplasm. Probably, a reduced cytoplasm counteracts a remarkable trait of CMR12a, namely the tendency to form spontaneous GacS/GacA mutations. The Gac system is a major regulator of secondary metabolism, including phenazine production, which has been described before to be susceptible to spontaneous mutations resulting in phenotypic switches called phase variation. It was shown that a gac mutant has a severely reduced cytoplasm, and that the stability of the gac locus in CMR12a possibly depends on the reduction status of the cytoplasm. Therefore, different frequencies of Gac-mutated CMR12a cultures were observed under aerobic and microaerobic conditions. Moreover, since phenazines are involved in maintaining the appropriate redox conditions of the cytoplasm, different frequencies of Gac-mutated cultures of CMR12a mutants with altered phenazine production (absence and overproduction of phenazines) were observed. The overproduction of phenazines in mutants in cytochrome o ubiquinol oxidase (CMR12a-PCyo) and in phosphoenolpyruvate phospho-transferase (CMR12a-PtsP) is thought to be a reaction of the cell against the reduction of the cytoplasm caused by the mutation. Possible regulatory mechanisms involved in this fine-tuning of phenazine production are considered. 
This work has provided more insight into the tropical biocontrol strain Pseudomonas sp. CMR12a in many aspects. The genome revealed special gene clusters and a novel quorum sensing system, which might ultimately be the result of special adaptations to the environment. The physiological relevance of phenazine reduction in Pseudomonas sp.CMR12a has been explored and may be contributing to rhizosphere competence of phenazine producing strains in general.},
  author       = {De Maeyer, Katrien},
  isbn         = {9789059894938},
  keyword      = {biological control,phenazines,pseudomonas species,quorum sensing},
  language     = {eng},
  pages        = {III, 217},
  publisher    = {Ghent University. Faculty of Bioscience Engineering},
  school       = {Ghent University},
  title        = {The versatile role of quorum sensing and phenazines in the biocontrol strain Pseudomonas sp. CMR12a},
  year         = {2012},
}

Chicago
De Maeyer, Katrien. 2012. “The Versatile Role of Quorum Sensing and Phenazines in the Biocontrol Strain Pseudomonas Sp. CMR12a”. Ghent, Belgium: Ghent University. Faculty of Bioscience Engineering.
APA
De Maeyer, K. (2012). The versatile role of quorum sensing and phenazines in the biocontrol strain Pseudomonas sp. CMR12a. Ghent University. Faculty of Bioscience Engineering, Ghent, Belgium.
Vancouver
1.
De Maeyer K. The versatile role of quorum sensing and phenazines in the biocontrol strain Pseudomonas sp. CMR12a. [Ghent, Belgium]: Ghent University. Faculty of Bioscience Engineering; 2012.
MLA
De Maeyer, Katrien. “The Versatile Role of Quorum Sensing and Phenazines in the Biocontrol Strain Pseudomonas Sp. CMR12a.” 2012 : n. pag. Print.