Spontaneous Gac Mutants of Pseudomonas Biological Control Strains: Cheaters or Mutualists?

Department of Ecology and Evolutionary Biology, University of Arizona, Tucson, Arizona, USA.
Applied and Environmental Microbiology (Impact Factor: 3.67). 08/2011; 77(20):7227-35. DOI: 10.1128/AEM.00679-11
Source: PubMed


Bacteria rely on a range of extracellular metabolites to suppress competitors, gain access to resources, and exploit plant or animal hosts. The GacS/GacA two-component regulatory system positively controls the expression of many of these beneficial external products in pseudomonad bacteria. Natural populations often contain variants with defective Gac systems that do not produce most external products. These mutants benefit from a decreased metabolic load but do not appear to displace the wild type in nature. How could natural selection maintain the wild type in the presence of a mutant with enhanced growth? One hypothesis is that Gac mutants are "cheaters" that do not contribute to the public good, favored within groups but selected against between groups, as groups containing more mutants lose access to ecologically important external products. An alternative hypothesis is that Gac mutants have a mutualistic interaction with the wild type, so that each variant benefits by the presence of the other. In the biocontrol bacterium Pseudomonas chlororaphis strain 30-84, Gac mutants do not produce phenazines, which suppress competitor growth and are critical for biofilm formation. Here, we test the predictions of these alternative hypotheses by quantifying interactions between the wild type and the phenazine- and biofilm-deficient Gac mutant within growing biofilms. We find evidence that the wild type and Gac mutants interact mutualistically in the biofilm context, whereas a phenazine-defective structural mutant does not. Our results suggest that the persistence of alternative Gac phenotypes may be due to the stabilizing role of local mutualistic interactions.

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Available from: William W Driscoll, Oct 07, 2015
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    • "A wealth of research has focused on understanding the factors that promote public goods cooperation in the light of the prevalent risk of cheater exploitation. This research has demonstrated that the evolutionary success of microbial cooperation depends on many factors, including strain frequency (Gilbert et al., 2007; Ross-Gillespie et al., 2007; Gore et al., 2009), cell density (Greig & Travisano, 2004; Ross-Gillespie et al., 2009), resource availability (Brockhurst et al., 2008; K€ ummerli et al., 2009b), cell dispersal (Chao & Levin, 1981; MacLean & Brandon, 2008; K€ ummerli et al., 2009a; Julou et al., 2013; Refardt et al., 2013), diffusion of public goods (K€ ummerli et al., 2009a; Le Gac & Doebeli, 2010), durability of public goods (K€ ummerli & Brown, 2010), regulatory mechanisms that allow an optimal timing of public goods production (K€ ummerli & Brown, 2010; Xavier et al., 2011; Darch et al., 2012) and pleiotropic effects emerging from the genetic architecture of social traits (Driscoll et al., 2011; Dandekar et al., 2012). While this body of experimental work, as a whole, demonstrates the complexity of microbial public goods cooperation, individual studies usually focused on the impact of a single factor at the time (see Brockhurst et al., 2010 as an exception). "
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    ABSTRACT: Microbial cooperation typically consists in the sharing of secreted metabolites (referred to as public goods) within the community. Although public goods generally promote population growth, they are also vulnerable to exploitation by cheating mutants, which no longer contribute, but still benefit from the public goods produced by others. Although previous studies have identified a number of key factors that prevent the spreading of cheaters, little is known about how these factors interact and jointly shape the evolution of microbial cooperation. Here, we address this issue by investigating the interaction effects of cell diffusion, cell density, public good diffusion and durability (factors known to individually influence costs and benefits of public goods production) on selection for cooperation. To be able to quantify these effects across a wide parameter space, we developed an individual-based simulation platform, consisting of digital cooperator and cheater bacteria inhabiting a finite two-dimensional continuous toroidal surface. Our simulations, which closely mimic microbial microcolony growth, revealed that: (i) either reduced cell diffusion (which keeps cooperators together) or reduced public good diffusion (which keeps the public goods closer to the producer) is not only essential but also sufficient for cooperation to be promoted; (ii) the sign of selection for or against cooperation can change as a function of cell density and in interaction with diffusion parameters; and (iii) increased public goods durability has opposing effects on the evolution of cooperation depending on the level of cell and public good diffusion. Our work highlights that interactions between key parameters of public goods cooperation give rise to complex fitness landscapes, a finding that calls for multifactorial approaches when studying microbial cooperation in natural systems.
    Journal of Evolutionary Biology 06/2014; 27(9). DOI:10.1111/jeb.12437 · 3.23 Impact Factor
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    • "The PA23 wild type consistently reached a higher OD600 in stationary phase compared to PA23-443 (Figure 4). A similar altered pattern of growth has been observed for gacS mutants of PA23 and 30–84 which exhibit a shorter lag phase and earlier entry into logarithmic growth phase [4,29]. LTTRs have previously been implicated in the regulation of cellular growth factors. "
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    ABSTRACT: Pseudomonas chlororaphis strain PA23 is a biocontrol agent capable of suppressing the fungal pathogen Sclerotinia sclerotiorum. This bacterium produces the antibiotics phenazine and pyrrolnitrin together with other metabolites believed to contribute to biocontrol. A mutant no longer capable of inhibiting fungal growth was identified harboring a transposon insertion in a gene encoding a LysR-type transcriptional regulator (LTTR), designated ptrA (Pseudomonas transcriptional regulator). Isobaric tag for relative and absolute quantitation (iTRAQ) based protein analysis was used to reveal changes in protein expression patterns in the ptrA mutant compared to the PA23 wild type. Relative abundance profiles showed 59 differentially-expressed proteins in the ptrA mutant, which could be classified into 16 clusters of orthologous groups (COGs) based on their predicted functions. The largest COG category was the unknown function group, suggesting that many yet-to-be identified proteins are involved in the loss of fungal activity. In the secondary metabolite biosynthesis, transport and catabolism COG, seven proteins associated with phenazine biosynthesis and chitinase production were downregulated in the mutant. Phenotypic assays confirmed the loss of phenazines and chitinase activity. Upregulated proteins included a lipoprotein involved in iron transport, a flagellin and hook-associated protein and four proteins categorized into the translation, ribosome structure and biogenesis COG. Phenotypic analysis revealed that the mutant exhibited increased siderophore production and flagellar motility and an altered growth profile, supporting the proteomic findings. PtrA is a novel LTTR that is essential for PA23 fungal antagonism. Differential protein expression was observed across 16 COG categories suggesting PtrA is functioning as a global transcriptional regulator. Changes in protein expression were confirmed by phenotypic assays that showed reduced phenazine and chitinase expression, elevated flagellar motility and siderophore production, as well as early entrance into log phase growth.
    BMC Microbiology 04/2014; 14(1):94. DOI:10.1186/1471-2180-14-94 · 2.73 Impact Factor
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    • "The biofilms produced by the gacS mutant on the minimal medium were generated without phenazine formation. Phenazines are cited to promote attachment of P. chlororaphis 30-84 in the initial stages of biofilm formation (Maddula et al. 2008; Driscoll et al. 2011) and to provide an electron sink under low oxygen conditions (Ramos et al. 2010). Studies in the phenazine-inducing AB medium confirmed that the gacS mutant of P. chlororaphis O6 was impaired in AHSL formation. "
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    ABSTRACT: An aggressive root colonizer, Pseudomonas chlororaphis O6 produces various secondary metabolites that impact plant health. The sensor kinase GacS is a key regulator of the expression of biocontrol-related traits. Biofilm formation is one such trait because of its role in root surface colonization. This paper focuses on the effects of carbon source on biofilm formation. In comparison with the wild type, a gacS mutant formed biofilms at a reduced level with sucrose as the major carbon source but at much higher level with mannitol in the defined medium. Biofilm formation by the gacS mutant occurred without phenazine production and in the absence of normal levels of acyl homoserine lactones, which promote biofilms with other pseudomonads. Colonization of tomato roots was similar for the wild type and gacS mutant, showing that any differences in biofilm formation in the rhizosphere were not of consequence under the tested conditions. The reduced ability of the gacS mutant to induce systemic resistance against tomato leaf mold and tomato gray mold was consistent with a lack of production of effectors, such as phenazines. These results demonstrated plasticity in biofilm formation and root colonization in the rhizosphere by a beneficial pseudomonad.
    Canadian Journal of Microbiology 03/2014; 60(3):133-8. DOI:10.1139/cjm-2013-0736 · 1.22 Impact Factor
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