Oxygen-Dependent Globin Coupled Sensor Signaling Modulates

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Oxygen-Dependent Globin Coupled Sensor Signaling Modulates Motility and Virulence of the Plant Pathogen Pectobacterium carotovorum Justin L. Burns, Parth B. Jariwala, Shannon Rivera, Benjamin M. Fontaine, Laura Briggs, and Emily E. Weinert ACS Chem. Biol., Just Accepted Manuscript • DOI: 10.1021/acschembio.7b00380 • Publication Date (Web): 14 Jun 2017 Downloaded from http://pubs.acs.org on June 16, 2017

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ACS Chemical Biology

Oxygen-Dependent Globin Coupled Sensor Signaling Modulates Motility and Virulence of the Plant Pathogen Pectobacterium carotovorum

Justin L. Burns§, Parth B. Jariwala§, Shannon Rivera, Benjamin M. Fontaine#, Laura Briggs#, and Emily E. Weinert*

Department of Chemistry, Emory University, 1515 Dickey Drive, Atlanta, GA 30322; 404-712-6865, [email protected]

§

These authors contributed equally to this work.

#

These authors contributed equally to this work.

* Corresponding author

ACS Paragon Plus Environment


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Abstract Bacterial pathogens utilize numerous signals to identify the presence of their host and coordinate changes in gene expression that allow for infection. Within plant pathogens, these signals typically include small molecules and/or proteins from their plant hosts and bacterial quorum sensing molecules to ensure sufficient bacterial cell density for successful infection. In addition, bacteria use environmental signals to identify conditions when the host defenses are weakened and potentially to signal entry into an appropriate host/niche for infection. A globin coupled sensor protein (GCS), termed PccGCS, within the soft rot bacterium Pectobacterium carotovorum ssp. carotovorum WPP14 has been identified as an O2 sensor and demonstrated to alter virulence factor excretion and control motility, with deletion of PccGCS resulting in decreased rotting of a potato host. Using small molecules that modulate bacterial growth and quorum sensing, PccGCS signaling also has been shown to modulate quorum sensing pathways, resulting in the PccGCS deletion strain being more sensitive to plant-derived phenolic acids, which can function as quorum sensing inhibitors, and exhibiting increased N-acylhomoserine lactone (AHL) production. These findings highlight a role for GCS proteins in controlling key O2dependent phenotypes of pathogenic bacteria and suggest that modulating GCS signaling to limit P. carotovorum motility may provide a means to decrease rotting of plant hosts.

INTRODUCTION Soft rot bacteria are a family of plant pathogens that infect their hosts by excreting enzymes that degrade plant cell walls, leading to characteristic rotting.1, 2 Bacterial soft rot is a widespread disease of cash crops around the world and is estimated to cause


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losses of hundreds of millions of dollars annually.1, 2 Gram-negative bacteria from the genera Erwinia, Pectobacterium (formerly Erwinia), and Dickeya are commonly responsible for soft rot. These bacteria are known to sense plant phenolic acids and quorum sensing molecules, such as N-acylhomoserine lactones (AHL), and coordinate attack on their hosts by releasing plant cell wall degrading enzymes that lead to rotting.1, 2 Many of the genes encoding virulence factors have been identified and their role in pathogenesis investigated. These include the excreted enzymes that are responsible for degrading the plant host, including proteases, pectinases, pectate lyases, and cellulases.1-4 However, many of the molecular signals and sensor proteins controlling virulence are unknown and effective control of these plant pathogens has remained elusive.1, 2, 5-9

In terms of environmental signals, oxygen (O2) concentration has been demonstrated to affect the pathogenicity of soft rot bacteria,10 but the mechanism by which O2 is sensed and the signal transmitted within Pectobacterium has yet to be established. Decreased O2 levels, relative to air, are often found when fields are flooded or crops are stored without adequate ventilation

11, 12

and lead to increased rotting. This is likely in part because the

plant wound response requires O2 for generation of reactive oxygen species to crosslink proteins and sugars within their cell walls to isolate the infection, as well as kill the invading microbes.13-15 Therefore, at low O2 levels, the plants are unable to adequately repel microbial invasions. In addition, virulence gene expression in Erwinia is increased in low O2 conditions,5, 16 suggesting that the bacteria may have evolved to take advantage of conditions that result in decreased host wound response.



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Potential regulators of O2-dependent phenotypes in soft rot bacteria are globin coupled sensor (GCS) proteins, which are a family of heme proteins that consist of a N-terminal sensor globin domain linked through a middle domain to a variety of output domains, such as methyl accepting chemotaxis proteins (MCP), diguanylate cyclases, phosphodiesterases, STAS domains, and kinases.17-23 GCS proteins are widely distributed throughout bacteria, as well as in a number of archaea, and are represented within the genomes of environmental bacteria, plant pathogens, and human pathogens, suggesting widespread importance in controlling O2-dependent bacterial phenotypes.23 Ligand binding to the heme within the sensor globin domain of GCSs results in altered activity of the output domain, leading to phenotypes such as aerotaxis and biofilm formation.18, 20, 22, 24-27

While studies have provided information about the enzymatic functions of these

proteins, little is known about the in vivo effects of GCS signaling.

A single GCS is encoded in the Pectobacterium carotovorum genome (termed PccGCS) and it is a soluble, cytoplasmic protein that contains a diguanylate cyclase (DGC) output domain, which is the enzymatic domain responsible for synthesizing c-di-GMP, a bacterial second messenger that regulates biofilm formation.28-30 In vitro characterization of PccGCS has demonstrated that gaseous ligand binding to the globin domain heme regulates cyclase activity.18 Binding of O2 to the heme results in increased cyclic di-GMP production, whereas the FeII unligated state results in basal cyclase activity,18 suggesting that PccGCS may be an in vivo sensor regulating O2-dependent biofilm formation and virulence. Furthermore, it has been shown that other GCS proteins, including those from Escherichia coli, Bordetella pertussis, Shewanella putrefaciens, and Pectobacterium


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atrosepticum,20,

22, 25, 31, 32

affect c-di-GMP levels and biofilm formation in vivo.

Therefore, O2-dependent PccGCS signaling in P. carotovorum was investigated to determine the effects on growth, downstream pathways, and virulence within soft rot bacteria.

RESULTS AND DISCUSSION PccGCS does not alter P. carotovorum growth Pectobacterium carotovorum ssp. carotovorum WPP14 (PccWPP14), originally isolated from potato fields in Wisconsin,33 was used in this study due to its previously characterized virulence in potatoes and the available genome sequence. The gene encoding PccGCS in PccWPP14 (accession: ZP_03830919) is located in an operon with a number of genes involved in chemotaxis, but genes putatively encoding c-di-GMP binding proteins are not found within the operon. As expected based on work in other Gram-negative bacteria (E. coli,34 S. putrefaciens,25 and B. pertussis22), disruption of the GCS gene (∆GCS PccWPP14) was not lethal, confirming that PccGCS is not an essential gene.

As PccGCS is predicted to serve as an O2 sensor and therefore might be controlling O2dependent metabolism, the effects of deleting PccGCS on growth of PccWPP14 were measured. No differences in growth rate between WT and ∆GCS PccWPP14 were observed when the strains were grown under aerobic conditions in either minimal media (M9) (Figure S1) or minimal media plus lettuce leaf lysate (M9+LLL) (Figure 1), which was added to mimic growth in a plant host. Furthermore, growth in M9+LLL under



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anaerobic conditions did not differ between WT and ∆GCS PccWPP14 (Figure 1A). Taken together, these data suggest that GCS signaling within Pectobacterium is required only under certain conditions and is not needed for robust growth in liquid media.

Plants are known to activate molecular oxygen to generate reactive oxygen species (ROS), which can crosslink the plant cell wall to isolate the infection and to kill the invading microbes.14 Therefore, PccGCS signaling potentially could be acting as a ROS sensor, thereby monitoring cellular redox state, in addition to sensing O2 levels. To determine if GCS signaling affects the ability of PccWPP14 to respond to ROS, WT and ∆GCS PccWPP14 were either grown in the presence of H2O2 (Figure 1B) or challenged with H2O2 upon entering mid-log phase (Figure 1C). When WT and ∆GCS PccWPP14 were grown in M9+LLL with H2O2, both strains grew at the same rate regardless of the absence of presence of H2O2, even up to concentrations of 1 mM H2O2 in the media. Growth in M9 yielded similar results with no difference between the WT and ∆GCS strains, but both strains exhibited a long lag phase even at 100 µM H2O2 (Figure S1), highlighting the importance of plant-derived small molecules in allowing PccWPP14 to tolerate ROS. Furthermore, challenging WT and ∆GCS PccWPP14 with H2O2 during mid-log phase resulted in similar growth behavior (Figure 1C), suggesting that PccGCS is not involved in sensing and/or responding to plant-derived ROS.

Virulence factor and AHL production are modulated by PccGCS Based on in vitro studies that demonstrated that PccGCS synthesizes c-di-GMP and that catalysis is dependent on the ligation state of the heme within the globin domain,18 we


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hypothesized that O2-dependent c-di-GMP production by PccGCS may control phenotypes involved in virulence within a plant host. As it previously has been established that virulence of soft rot bacteria depends on O2 levels

5, 10, 16

virulence factors expression is typically decreased within a biofilm,9,

35

and that

c-di-GMP

produced by PccGCS was hypothesized to be at least partially responsible for controlling downstream pathways and phenotypes in an O2-dependent manner. Therefore, as c-diGMP is a major regulator of biofilm production,28, 30 the effect of deleting PccGCS on biofilm formation was investigated. When using Congo Red staining,36 PccWPP14 ∆GCS strain exhibited decreased biofilm formation when grown aerobically on plates, as compared to WT PccWPP14 (Figure S2). Furthermore, although PccWPP14 did not generate robust biofilms, low levels of biofilm formation could be quantified (Figure 2). Under both aerobic and anaerobic conditions, ∆GCS PccWPP14 exhibited decreased biofilm formation, as compared to WT PccWPP14. Given that PccGCS exhibits a modest (~2.5-fold) change in cyclase activity upon O2 binding, PccGCS is active even in the absence of O2 (FeII-O2 kcat = 0.73 min-1; FeII kcat = 0.29 min-1),18 and that PccWPP14 has multiple diguanylate cyclases encoded within the genome, it is not surprising that robust O2-dependent changes were not observed. However, these data do demonstrate that PccGCS is active in vivo and affects P. carotovorum biofilm formation.

Having confirmed that PccGCS is active and can affect Pectobacterium biofilm formation in vivo (Figure 2), the role(s) in virulence were investigated. Typically, biofilm formation and virulence are inversely correlated, with increased biofilm formation leading to decreased excretion of virulence factors.9, 35 Therefore, the decrease in biofilm



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formation for the ∆GCS PccWPP14 strain suggested that there might be a concomitant change in virulence factor production. Soft rot bacteria excrete virulence factors that comprise four main classes of enzymes: proteases (Prt), cellulases (Cel), pectate lyases (Pel), and endopolygalacturonases (Peh, also hydrolyze pectate).1-3 These enzymes are used to degrade the plant host, releasing nutrients and causing the characteristic rotting. Furthermore, overexpression of a putative diguanylate cyclase-containing GCS in P. atrosepticum previously was shown to alter virulence factor activity (increased Cel activity, decreased Pel and Prt activity).32 Therefore, dependence of virulence factor excretion on PccGCS and O2 was interrogated by quantifying exoenzyme activity. When grown aerobically, ∆GCS PccWPP14 exhibited lower levels (~15% decrease) of excreted enzyme activity for all four enzyme classes, as compared with WT PccWPP14 (Figure 3A). However, when PccWPP14 WT and ∆GCS strains were grown anaerobically, only endopolygalacturonase (Peh) and cellulose (Cel) activity were observable, with slightly increased Peh activity for ∆GCS PccWPP14 and nearly identical Cel activity for both strains (Figure 3B). These data demonstrate the importance of GCS signaling pathways in controlling aerobic virulence factor production and provide evidence of a role for GCS proteins in bacterial soft rot.

The decreased virulence factor excretion observed for aerobically grown PccWPP14 ∆GCS, as compared to WT PccWPP14, suggested that O2-dependent GCS signaling might intersect with quorum sensing pathways.37, 38 Quorum sensing (QS), cell-to-cell bacterial communication utilizing excreted small molecules, such as N-acylhomoserine lactones (AHL), previously has been shown to be a master regulator of P. carotovorum


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virulence-related pathways and has been shown to control diverse pathways in other Gram-negative bacteria, including growth, motility, and metabolism.1, 5, 6, 37, 39 To test this hypothesis, the effects of small molecule QS modulators on growth and motility of WT and ∆GCS PccWPP14 were investigated. Recent studies have demonstrated that plantderived phenolic acids can modulate Pectobacterium QS pathways. The phenolic acids exhibit anti-microbial activity at high concentrations, but at sub-inhibitory concentrations the compounds decrease Pectobacterium AHL production, QS gene expression, and virulence.40, 41 Therefore, salicyclic acid and cinnamic acid, two of the identified phenolic acids that are prevalent in potatoes, and a native Pectobacterium AHL, N-(3-oxo)hexanoyl L-homoserine lactone (1) (Figure 4A),38, 42 were chosen to use as tools to probe possible interplay between PccGCS signaling and quorum sensing pathways.

Growth in the presence of both salicylic acid and cinnamic acid (≥2 mM) resulted in similar growth rates for WT and ∆GCS PccWPP14 through mid-log phase. However, ∆GCS PccWPP14 could not attain similar stationary phase cell densities to WT PccWPP14 in the presence of high concentrations (2–3 mM) of salicyclic acid or cinnamic acid (Figure 5A and 5B, respectively). These data suggest that ∆GCS PccWPP14 is more sensitive to disruption of AHL-dependent pathways and/or might have altered levels of AHL production, supporting intercommunication between PccGCS signaling and quorum sensing pathways.

To further investigate potential links link between PccGCS signaling and quorum sensing pathways, production of AHL (1) by WT and ∆GCS PccWPP14 was quantified.



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Surprisingly, ∆GCS PccWPP14 excreted ~2-fold more AHL (1) than WT PccWPP14 (Figure 4B). Typically, increased extracellular AHL levels correlate with increased intracellular levels, as AHL are membrane permerable, and result in increased virulence and exoenzyme excretion.37, 38, 43 However, while ∆GCS PccWPP14 exhibits increased extracellular levels of AHL (1), intracellular levels of AHL (1) in ∆GCS PccWPP14 were found to be within error of the levels in WT PccWPP14. Therefore, the observations that ∆GCS PccWPP14 exhibits decreased virulence and exoenzyme excretion, but increased total AHL production while maintaining intracellular AHL levels, suggests that PccGCS may be regulating expression and/or activity of efflux pumps. Previous studies on Pseudomonas aeruginosa demonstrated that homoserine lactone levels are controlled by both diffusion and active efflux.44, 45 The P. aeruginosa AHL, N-(3-oxo)-dodecanoyl-Lhomoserine lactone, was shown to be a substrate for the MexAB-OprM multidrug efflux system, which regulated AHL levels and AHL-dependent downstream phenotypes. Furthermore, a gene within the PccGCS operon is predicted to encode a drug/metabolite transporter (ZP_03830922), suggesting that PccGCS O2-dependent c-di-GMP production might control transporter activity. Taken together, these studies suggest that PccGCS both increases AHL production, either through up-regulation of AHL synthase activity or expression levels, and increases efflux of AHL (1), which limits activation of AHLdependent QS pathways.

PccGCS Regulates O2-Dependent Motility Localization of PccGCS in an operon with proteins putatively involved in chemotaxis, including a methyl accepting chemotaxis proteins, CheW (signal transduction protein),


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CheA (sensor histidine kinase), Mot A and B (flagellar motor proteins), and FlhC and FlhD (flagellar transcriptional activators), suggested a possible role for PccGCS in regulating P. carotorum motility. Under aerobic conditions, ∆GCS PccWPP14 was found to exhibit >2-fold decreased motility, as compared to WT PccWPP14, while there is no statistical difference under anaerobic conditions (Figure 6A and 6B). These PccGCS- and O2- dependent changes in motility should decrease the ability of ∆GCS PccWPP14 to move through infiltrated plant tissue under aerobic conditions, limiting the range of rot within a plant host.

As QS pathways previously have been linked to motility5 and Erwinia chrysanthemi exhibited increased motility in the absence of AHL production,43 the interplay of PccGCS-regulated motility and QS pathways was investigated using AHL (1), the phenolic acid QS modulators (salicyclic acid and cinnamic acid), and a quorum sensing inhibitor46 (QSI, (2); Fig. 4A). Despite the lack of increase in intracellular AHL concentration, increased extracellular levels of AHL (1) potentially could affect motility of ∆GCS PccWPP14 through extracellular interactions. However, incubation of WT and ∆GCS PccWPP14 with AHL (1) did not result in any motility changes, as compared with WT and ∆GCS PccWPP14 assayed in the absence of (1) (Figure 6C, Figure S4), demonstrating that increased levels of AHL (1) likely are not responsible for increased motility of ∆GCS PccWPP14. A small decrease in motility (~1.4-fold) was observed for WT PccWPP14 in the presence of salicylic acid and cinnamic acid, which were previously demonstrated to decrease P. carotovorum AHL production. However, exposure of ∆GCS PccWPP14 to either salicylic acid or cinnamic acid (Figure 6D),



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resulted in nearly indistinguishable motility as compared to ∆GCS PccWPP14 in the absence of small molecule modulators. Furthermore, incubation with QSI (2), which previously was demonstrated to inhibit Pectobacterium QS signaling,46 did not result in motility changes of the WT or ∆GCS PccWPP14 (Figure 6E). These results demonstrate that the decreased motility observed for ∆GCS PccWPP14 is due to PccGCS regulation of chemotaxis-related proteins, rather than an effect of differences in QS pathways.

Recent studies identifying direct interactions between diguanylate cyclases and motility machinery47-49 suggested that PccGCS regulation of O2-dependent motility could be due to protein-protein interactions with motility-related proteins in the same operon. Therefore, interactions between PccGCS and other P. carotovorum proteins were identified by affinity pull-down assays followed by mass spectrometry analysis. The CheW-like protein (ZP_03830929) in the PccGCS operon was identified in the experiments (100% sequence coverage), suggesting a robust interaction between the two proteins (Figure S3). CheW proteins typically serve to couple a chemotaxis transducers and CheA histidine kinase, leading to signal-dependent motility.50 Indeed, peptides corresponding to CheA (ZP_03830928, 73% sequence coverage) and TarH (ZP_03830921, 66% sequence coverage) also were identified from the pull-down experiments, suggesting that PccGCS interacts with the entire signaling complex to regulate O2-dependent chemotaxis.

O2-Dependent Rotting of a Plant Host


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To investigate the cumulative effects PccGCS-dependent changes on P. carotovorum virulence, potato tubers were chosen as the model plant host because PccWPP14 was isolated from Wisconsin potato fields, soft rot bacterial infections of potato tubers have been extensively characterized, and methods to quantify potato tuber rotting have previously been described.6,

7, 10, 33

Furthermore, Pectobacterium species are major

contributors to soft rot that negatively affects crop yields,1,

2

suggesting that insight

gained from these studies may be applicable to designing new inhibitors of soft rot in agriculturally important crops. Therefore, PccWPP14 WT and ∆GCS strains were injected into potato tubers and incubated aerobically or in an anaerobic chamber to determine the effects of both PccGCS and O2 on virulence.

As previously observed,10,

16

rotting was significantly increased when PccWPP14-

infected potatoes were incubated under anaerobic conditions, as compared with aerobic incubation (Figure 7A). Quantification of rotted tissue demonstrated that aerobic incubation led to dramatic differences in rotting within the potato (Figure 7, S5). While there was significant potato-to-potato variability in the amount of rotted tissue (Figure 7A), likely due to differences in the levels of anti-microbial and attractive compounds within tubers harvested from different locations,51-53 ∆GCS PccWPP14 always yielded decreased rotting, as compared to WT PccWPP14 (Figure 7B). Despite the differences in aerobic rotting, WT and ∆GCS PccWPP14 exhibited similar growth within the potato (Figure 7C). In contrast, following anaerobic incubation, there was no significant difference between WT and ∆GCS PccWPP14 in terms of rotting or growth within the potato (Figure 7A, right panel) and no observable trend within each potato. These data



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demonstrate that PccGCS affects P. carotovorum rotting of the potato host. As the changes in virulence factor excretion observed for ∆GCS PccWPP14 are modest (~15% decrease), the defect in rotting within potatoes likely also is due to the decreased motility of ∆GCS PccWPP14, limiting the diffusion range of the excreted virulence factors.

In summary, we have demonstrated that PccGCS serves as an O2 sensor within the soft rot bacterium P. carotovorum. These studies demonstrate for the first time, to our knowledge, a role for globin coupled sensor proteins in controlling O2-dependent virulence of a pathogenic bacterium (Figure 8). Our work suggests broader roles for O2 sensing by GCS proteins in controlling downstream bacterial phenotypes, including through direct protein-protein interactions with motility machinery. Given that quorum sensing previously was shown to control virulence factor excretion1,

3, 37, 39

and that

deletion of PccGCS increases AHL production, studies are underway to investigate how GCS downstream signaling events intersect with QS pathways or can directly control gene expression to modulate activation of P. carotovorum virulence factor production. In addition, due to the agricultural importance of soft rot infections, modulating GCS sensing and/or signaling may provide novel methods to decrease virulence of soft rot bacteria.

METHODS All detailed methods can be found in the Supporting Information.


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Acknowledgments This work was supported by US National Science Foundation grant CHE 1352040 (E.E.W.) and a University Research Committee Grant from Emory University (E.E.W). The authors thank A. Charkowski (University of Wisconsin, Madison) for the P. carotovorum WPP14 WT strain, F. Strobel of the Emory Chemistry Mass Spectrometry Center for assistance with mass spectrometry experiments, J. Walker for experimental assistance, and members of the Weinert group for helpful comments.

Author Contributions JLB, PBJ, BMF, SR, LB, and EEW designed the study and performed all experiments. JLB, PBJ, BMF, SR, LB, and EEW analyzed the data. EEW wrote the manuscript.

Supporting Information. Supporting Information Available: This material is available free of charge via the Internet. P. carotovorum WT and ∆GCS growth in M9 media (Figure S1), quantitative biofilm comparison (Figure S2), mass spectra and fragmentation of CheW peptides (Figure S3), motility +/- AHL after 48h (Figure S4), representative results from a potato assay (Figure S5), and detailed Methods can be found in the Supporting Information.

[1] Charkowski, A. O. (2009) Decaying signals: will understanding bacterial-plant communications lead to control of soft rot?, Curr. Opin. Biotechnol. 20, 178–184. [2] Toth, I. K., and Birch, P. R. (2005) Rotting softly and stealthily, Curr. Opin. Plant Biol. 8, 424–429. [3] Chatterjee, A., Cui, Y., Liu, Y., Dumenyo, C. K., and Chatterjee, A. K. (1995) Inactivation of rsmA leads to overproduction of extracellular pectinases,



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Figure 1. Comparison of growth of P. carotovorum WPP14 WT (black) and ∆GCS (red) strains. a.) Growth curves under aerobic (solid lines) and anaerobic (dashed lines) conditions. b.) Growth in the presence of varying concentrations of H2O2. c.) Growth following challenge with varying concentrations of H2O2 (0–2 mM; H2O2 addition marked by an arrow).

Figure 2. Biofilm quantification of P. carotovorum WPP14 WT (black) and ∆GCS (red) strains grown under aerobic and anaerobic conditions. Differences between data for all samples are statistically significant as assessed by an unpaired t-test (aerobic, P < 0.05; anaerobic, P < 0.02).



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Figure 3. Exoenzyme assays for virulence factors excreted from P. carotovorum WPP14 WT (black) and ∆GCS (red) strains. Plate clearance zones were standardized to cell density when excreted enzymes were isolated. a.) Excretion when strains were grown under aerobic conditions. All differences are statistically significant as assessed by an unpaired t-test (Peh P < 0.008; Pel P < 0.0015; Prt P < 0.0015; Cel P < 0.009). b.) Excretion when strains were grown under anaerobic conditions.


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Figure 4. Quorum sensing regulation. a.) Structures of quorum sensing modulators. b.) AHL quantification of WT (black) and ∆GCS (red) PccWPP14 strains.



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Figure 5. Comparison of growth of P. carotovorum WPP14 WT (black, solid lines) and ∆GCS (red, dashed lines) strains in the presence of varying concentrations of a.) salicylic acid and b.) cinnamic acid.


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Figure 6. Motility assays for P. carotovorum WPP14 WT (black) and ∆GCS (red) strains. a.) and b.) Motility of WPP14 WT (black) and ∆GCS (red) strains grown aerobically and anaerobically for a.) 24 hrs. and b.) 48 hrs. of incubation. c.), d.), and e.) Effect of QS modulators on the motility of PccWPP14 strains after 24 hrs. of incubation. c.) Effect of AHL (1) on motility of WT (black) and ∆GCS (red) PccWPP14 strains. d.) Effect of phenolic acids on motility of WT (black) and ∆GCS (red) PccWPP14 strains. Solid bars, control; diagonal stripe bars, salicyclic acid; empty bars, cinnamic acid. e.) Effect of QSI (2) on motility of WT (black) and ∆GCS (red) PccWPP14 strains. Solid bars, control; striped bars, QSI (2).


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Figure 7. Rotting and growth of P. carotovorum WPP14 WT and ∆GCS strains in Yukon Gold potatoes. a.) Quantified rotted tissue after aerobic (left) and anaerobic (right)



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incubation. Data from each potato is depicted in the same symbol/color for WT and ∆GCS. Whisker top: 90 percentile, box top: 75 percentile, box middle: 50 percentile, box bottom: 25 percentile and whisker bottom: 10 percentile. Differences between data for strains incubated aerobically are statistically significant as assessed by an unpaired t-test (P < 0.015). b.) Comparison of rotted tissue for WT (black) and ∆GCS (red) strains within each individual aerobic potato. c.) Number of colony forming units (CFU) recovered from WT (black) and ∆GCS (red) growth within potatoes.

Figure 8. O2-dependent PccGCS signaling in P. carotovorum. PccGCS directly interacts with motility-related proteins (TarH, CheA, CheW) to control O2-dependent motility. In addition, PccGCS signaling, likely through modulating c-di-GMP levels, regulates virulence factor excretion, AHL production (likely by AHL synthase ExpI), and internal/external AHL levels (potentially through regulation of an efflux pump).


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Figure 1. Comparison of growth of P. carotovorum WPP14 WT (black) and ∆GCS (red) strains. a.) Growth curves under aerobic (solid lines) and anaerobic (dashed lines) conditions. b.) Growth in the presence of varying concentrations of H2O2. c.) Growth following challenge with varying concentrations of H2O2 (0–2 mM; H2O2 addition marked by an arrow). 82x209mm (300 x 300 DPI)



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Figure 2. Biofilm quantification of P. carotovorum WPP14 WT (black) and ∆GCS (red) strains grown under aerobic and anaerobic conditions. Differences between data for all samples are statistically significant as assessed by an unpaired t-test (aerobic, P < 0.05; anaerobic, P < 0.02). 81x72mm (300 x 300 DPI)


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Figure 3. Exoenzyme assays for virulence factors excreted from P. carotovorum WPP14 WT (black) and ∆GCS (red) strains. Plate clearance zones were standardized to cell density when excreted enzymes were isolated. a.) Excretion when strains were grown under aerobic conditions. All differences are statistically significant as assessed by an unpaired t-test (Peh P < 0.008; Pel P < 0.0015; Prt P < 0.0015; Cel P < 0.009). b.) Excretion when strains were grown under anaerobic conditions. 83x143mm (300 x 300 DPI)



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Figure 4. Quorum sensing regulation. a.) Structures of quorum sensing modulators. b.) AHL quantification of WT (black) and ∆GCS (red) PccWPP14 strains. 86x139mm (300 x 300 DPI)


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Figure 5. Comparison of growth of P. carotovorum WPP14 WT (black, solid lines) and ∆GCS (red, dashed lines) strains in the presence of varying concentrations of a.) salicylic acid and b.) cinnamic acid. 84x145mm (300 x 300 DPI)



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Figure 6. Motility assays for P. carotovorum WPP14 WT (black) and ∆GCS (red) strains. a.) and b.) Motility of WPP14 WT (black) and ∆GCS (red) strains grown aerobically and anaerobically for a.) 24 hrs. and b.) 48 hrs. of incubation. c.), d.), and e.) Effect of QS modulators on the motility of PccWPP14 strains after 24 hrs. of incubation. c.) Effect of AHL (1) on motility of WT (black) and ∆GCS (red) PccWPP14 strains. d.) Effect of phenolic acids on motility of WT (black) and ∆GCS (red) PccWPP14 strains. Solid bars, control; diagonal stripe bars, salicyclic acid; empty bars, cinnamic acid. e.) Effect of QSI (2) on motility of WT (black) and ∆GCS (red) PccWPP14 strains. Solid bars, control; striped bars, QSI (2). 170x232mm (300 x 300 DPI)


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Figure 7. Rotting and growth of P. carotovorum WPP14 WT and ∆GCS strains in Yukon Gold potatoes. a.) Quantified rotted tissue after aerobic (left) and anaerobic (right) incubation. Data from each potato is depicted in the same symbol/color for WT and ∆GCS. Whisker top: 90 percentile, box top: 75 percentile, box middle: 50 percentile, box bottom: 25 percentile and whisker bottom: 10 percentile. Differences between data for strains incubated aerobically are statistically significant as assessed by an unpaired t-test (P < 0.015). b.) Comparison of rotted tissue for WT (black) and ∆GCS (red) strains within each individual aerobic potato. c.) Number of colony forming units (CFU) recovered from WT (black) and ∆GCS (red) growth within potatoes. 89x199mm (300 x 300 DPI)



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Figure 8. O2-dependent PccGCS signaling in P. carotovorum. PccGCS directly interacts with motility-related proteins (TarH, CheA, CheW) to control O2-dependent motility. In addition, PccGCS signaling, likely through modulating c-di-GMP levels, regulates virulence factor excretion, AHL production (likely by AHL synthase ExpI), and internal/external AHL levels (potentially through regulation of an efflux pump). 118x58mm (300 x 300 DPI)


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Table of Contents Graphic 79x36mm (300 x 300 DPI)


Oxygen-Dependent Globin Coupled Sensor Signaling Modulates

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