Iowa State University Capstones, Theses and Graduate Theses and Dissertations Dissertations
2021
Light as a global regulator of gene expression and an anticipatory signal for environmental water loss in Pseudomonas syringae
Bridget M. Hatfield Iowa State University
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Recommended Citation Hatfield, Bridget M., "Light as a global egulatr or of gene expression and an anticipatory signal for environmental water loss in Pseudomonas syringae" (2021). Graduate Theses and Dissertations. 18506. https://lib.dr.iastate.edu/etd/18506
This Dissertation is brought to you for free and open access by the Iowa State University Capstones, Theses and Dissertations at Iowa State University Digital Repository. It has been accepted for inclusion in Graduate Theses and Dissertations by an authorized administrator of Iowa State University Digital Repository. For more information, please contact [email protected]. Light as a global regulator of gene expression and an anticipatory signal for environmental water loss in Pseudomonas syringae
by
Bridget M. Hatfield
A dissertation submitted to the graduate faculty
in partial fulfillment of the requirements for the degree of
DOCTOR OF PHILOSOPHY
Major: Genetics
Program of Study Committee: Gwyn A. Beattie, Major Professor Mark L. Gleason Justin W. Walley Larry J. Halverson Kyaw J. Aung
The student author, whose presentation of the scholarship herein was approved by the program of study committee, is solely responsible for the content of this dissertation. The Graduate College will ensure this dissertation is globally accessible and will not permit alterations after a degree is conferred.
Iowa State University
Ames, Iowa
2021
Copyright © Bridget M. Hatfield, 2021. All rights reserved. ii
DEDICATION
To my husband, Kirk, your belief in me, encouragement, and love kept me going. To my daughter, Maeve, your never-ending joy always brightens my day. To my family, you have taught me to fight for my dreams and because of you I know that through hard work and persistence I can accomplish anything I set my mind to.
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TABLE OF CONTENTS
Page
ACKNOWLEDGMENTS ...... vi
ABSTRACT ...... vii
CHAPTER 1. GENERAL INTRODUCTION ...... 1 Dissertation Organization ...... 1 Literature Review ...... 1 Photosensory proteins are used to sense light ...... 2 Diverse photosensory proteins beyond those in Pss B728a ...... 3 Photosensory proteins in plants ...... 4 LOV proteins in microorganisms ...... 4 Phytochromes in microorganisms ...... 6 Photosensory proteins in Pseudomonas syringae ...... 7 P. syringae can survive in a wide range of environments ...... 8 Pss B728a as a plant pathogen ...... 9 Pss B728a strategies for coping with fluctuating water availability on leaves ...... 9 Goal of the thesis ...... 11 References ...... 11
CHAPTER 2. A BACTERIOPHYTOCHROME ORCHESTRATES TRANSCRIPTIONAL REPROGRAMMING IN RESPONSE TO MULTIPLE LIGHT WAVELENGTHS IN PSEUDOMONAS SYRINGAE B728a ...... 17 Abstract ...... 17 Introduction ...... 18 Materials and Methods ...... 20 Bacterial strains and growth conditions...... 20 Construction of a chloramphenicol resistant B728a strain...... 22 Construction of pET21a-bphOP1-His6X...... 22 Lighting conditions...... 22 Purification of BphP1 protein...... 23 Spectroscopic evaluation of BphP1 protein...... 23 Exposure of cells to light for RNAseq analysis...... 23 RNA extraction...... 24 Mapping the reads...... 25 RNAseq data analysis...... 25 Characterization of algD expression at various light intensities...... 26 Conjugation assays...... 26 Statistical analyses...... 27 Results ...... 27 Purified BphP1 responds to far-red, blue, and red light in vitro ...... 27 Light is a global signal for B728a gene expression...... 28 BphP1 regulates the majority of the light-responsive genes in B728a ...... 31 iv
Over-representation analyses identify functional groups that are strongly regulated by light ...... 34 Genes involved in osmotolerance are prominent among light- and BphP1-induced genes ...... 37 The light-dependent BphP1 regulon overlaps with the osmotic stress-dependent AlgU regulon ...... 40 BphP1 disproportionately induces genes that encode membrane-localized proteins...... 41 algD expression is sensitive to both quality and quantity of light ...... 42 BphP1 regulates conjugation in a light and wavelength specific manner ...... 43 Discussion ...... 47 Acknowledgements ...... 53 References ...... 54 Supplementary Data ...... 59
CHAPTER 3. PLANT-ASSOCIATED PSEUDOMONADS USE LIGHT AS AN ANTICIPATORY CUE TO ENHANCE OSMOTOLERANCE ...... 62 Abstract ...... 62 Introduction ...... 63 Methods ...... 67 Bacterial strains and growth conditions ...... 67 Light exposure setup ...... 68 Assessment of light impacts on bacterial osmotolerance based on optical density measurements ...... 68 Assessment of light impacts on bacterial osmotolerance based on cell viability ...... 69 Environmental monitoring preceding and following dawn ...... 70 Statistical Analysis ...... 71 Results ...... 72 Pre-exposure to far-red or red light enhances osmotolerance of Pss B728a...... 72 The magnitude of anticipatory light-enhanced osmotolerance is not strongly dependent on light-intensity ...... 74 The photosensory protein BphP1 is required for anticipatory light-enhanced osmotolerance in Pss B728a...... 75 Anticipatory light-enhanced osmotolerance requires the uptake and/or synthesis of osmoprotective compounds ...... 78 Pre-exposure to light does not improve cell viability immediately following osmotic upshift ...... 81 The onset of light at dawn precedes dew evaporation ...... 82 Anticipatory light-enhanced osmotolerance is conserved in several Pseudomonas spp...... 84 Discussion ...... 87 Acknowledgements ...... 92 References ...... 93
CHAPTER 4. GENERAL CONCLUSIONS ...... 98 BphR as a potential response receiver for BphP1 ...... 98 Anticipatory light-enhanced osmotolerance as a stress response strategy ...... 100 Anticipatory light-enhanced osmotolerance may not fully satisfy the criteria for an v
adaptive prediction response ...... 101 The integration of the BphP1 and AlgU regulon may occur via direct protein interactions in the membrane ...... 103 Plant photosensory proteins often function within multiprotein complexes, as predicted for BphP1 in B728a ...... 104 Insights from LOV- and phytochrome-mediated signal transduction mechanisms in microorganisms ...... 105 Future directions in identifying photosensory-regulator interactions in Pss B728a ...... 107 References ...... 108 vi
ACKNOWLEDGMENTS
I would like to thank my major professor, Dr. Gwyn A. Beattie, for her constant support and guidance as a mentor and for her contributions to my professional development. I would also like to thank my committee members Drs. Mark L. Gleason, Justin W. Walley, Larry J.
Halverson, and Kyaw J. Aung for their feedback and guidance.
I am grateful to the past and present colleagues in the Beattie and Halverson laboratories.
Your suggestions and comments during joint laboratory meetings are greatly appreciated.
Specifically, I would like to thank Dr. Regina S. McGrane for her patience in passing the knowledge she had developed during her graduate studies onto me so that I could make a smooth transition to working with a complicated system. I would also like to give a special thanks to Dr.
Olakunle I. Olawole, who came to work with a smile without fail - your happiness and eagerness to learn were inspiring. Additionally, I am grateful for the editing advice and writing instruction I have received from Dr. Breah LaSarre - your patience will forever be remembered.
Finally, I am thankful for the friendships I have made during my graduate studies. To all my classmates and colleagues, commiserating together over coursework and failed experiments allowed me to see I was not alone. Specifically, I am thankful to my friend Tesia, you have become a second sister to me and my time at Iowa State would not have been the same without you. vii
ABSTRACT
Light is a diel signal that is prevalent in many terrestrial environments. Light availability occurs in a cycle which temporally links light to a range of environmental fluctuations, such as temperature and ambient humidity. Therefore, light may be a powerful cue that photosensing organisms can exploit in an anticipatory manner to prepare for subsequent stresses. Photosensory proteins are widespread among plant-associated microbes, which is consistent with the requirement of light for plant growth. The leaf colonist and foliar pathogen Pseudomonas syringae pv. syringae (Pss) B728a contains genes for three photosensory proteins, one blue light- sensing LOV protein, and two red/far-red light-sensing bacteriophytochromes, BphP1 and
BphP2. Previous work has determined roles for LOV and BphP1 in influencing virulence, leaf colonization, and swarming motility in Pss B728a. Although LOV, BphP1, and BphP2 are histidine kinases, the target proteins that they phosphorylate have not yet been identified. To begin to identify cellular components that are influenced by these photosensory proteins, we conducted global transcriptome analyses characterizing the effects of individual wavelengths of light (far-red, red, and blue light) on wild-type Pss B728a and mutants lacking one or more photosensory proteins. We found that light is a global signal, initiating transcriptional changes in
31% of the genes in Pss B728a. While all tested wavelengths of light promoted changes in gene expression, far-red light influenced the most genes, suggesting a heightened sensitivity to far-red light. BphP1 was required for the majority of the transcriptional responses to all three wavelengths tested, consistent with the activation of purified BphP1 by all three wavelengths.
We demonstrated that light intensity, not just wavelength, impacts gene expression using algD as an indicator gene. Based on light- and BphP1-regulation of genes involved in conjugation, we performed functional tests and found that far-red light and BphP1 negatively regulate viii conjugation in Pss B728a. Light had an especially noticeable impact on genes involved in the response to osmotic stress, with these genes among those most strongly induced by light. A comparison of the light-responsive, BphP1-induced regulon with the previously characterized osmotic stress-responsive, AlgU-induced regulon showed a large, but not complete, overlap, highlighting that these two regulated pathways are integrated. We explored the possibility that light functions to help cells anticipate osmotic stress, finding that pre-exposure to far-red or red light enhances Pss B728a osmotolerance and that this light enhancement of osmotolerance requires BphP1. This anticipatory light-enhanced osmotolerance requires the extracytoplasmic function (ECF) sigma factor AlgU, which is a key regulator of osmotic stress response genes, as well as the AlgU-regulated functions of osmoprotectant transport (for experiments performed in the presence of the osmoprotectant choline) and compatible solute synthesis. Integration of the light- and osmotic stress-regulated pathways is further highlighted by BphP1 regulation of algU expression and AlgU regulation of expression of the photosensory gene lov. By monitoring the onset of light, dew accumulation, and surface water evaporation on sunlight-exposed surfaces at dawn, we provide evidence that exposure of terrestrial surfaces to light can precede rapid evaporative water loss during a natural diel cycle. This finding illustrates the potential biological benefit of anticipatory light-enhanced osmotolerance to leaf-surface bacteria. Lastly, we demonstrate that this anticipatory light-enhanced osmotolerance is likely a widespread phenomenon in plant-associated pseudomonads, and potentially within terrestrial microbes, although these have not yet been examined. Collectively, this work contains the most thorough investigation of light and photosensory proteins in a non-photosynthetic bacterium to date, and provides evidence of global regulation by a bacteriophytochrome, integration of responses to ix light and water stress, and a novel phytochrome-driven anticipatory stress response that modulates cellular fitness. 1
CHAPTER 1. GENERAL INTRODUCTION
Dissertation Organization
This dissertation is divided into four chapters. Chapter 1 contains a literature review of photosensory proteins and their impact on microbial ecology as well as the rational for conducting this research. Chapter 2 details the effects of various wavelengths of light and loss of photosensory proteins on global gene expression in P. syringae B728a and presents a characterization of light- and bacteriophytochrome-regulated functions, including conjugation.
Chapter 3 provides evidence that P. syringae B728a and other pseudomonads are capable of using light as an anticipatory signal to prepare for oncoming environmental stresses such as the water loss from leaf surfaces following dawn. Chapter 4 offers conclusions from this work as well as future directions that could be pursued.
Literature Review
Microorganisms can be found in almost every environment on the planet. These environments range from hot springs, oceans, deserts, and snow-peaked mountains to soils and leaf canopies (22; 53). Some of these environments are relatively constant, whereas others are highly dynamic. For instance, the phyllosphere is an environment that is particularly dynamic, with diel fluctuations in light, temperature, humidity, and water availability. Typically, microbes that are isolated from non-leaf habitats survive poorly on leaves, whereas those taken from leaves survive well when reintroduced (5; 34; 48). The ability of an organism to survive in a harsh environment or a highly fluctuating environment suggests that the organism has adaptations specific to that environment. Microbes in many terrestrial habitats are exposed to diel fluctuations in light, and these fluctuations likely co-occur with environmental changes such as the warming and drying of surfaces. Microbial habitats exposed to diel fluctuations include leaf 2 surfaces, terrestrial soils, rocks, and the surfaces of diurnal animals. Each of these terrestrial, surface environments are home to microbes that likely have adapted to the changing environmental conditions and changing light conditions specific to these locations.
The appearance of light at dawn may be a driving factor to aid in adaptive responses to diel environmental fluctuations. Some organisms contain photosensory proteins that allow them to respond to specific wavelengths of light. For this work the plant-associated bacterium
Pseudomonas syringae pv. syringae (Pss) strain B728a was used as a model organism. Pss
B728a is primarily an epiphyte and is therefore a good colonist of the leaf surface, suggesting that it can adapt to the fluctuating environment of the phyllosphere. Pss B728a contains three photosensory proteins, a blue light-sensing LOV protein and two red/far-red light-sensing bacteriophytochromes. Here we investigated how the presence of specific wavelengths of light and photosensory proteins impact gene expression in Pss B728a. Following the analysis of gene expression changes under specific light conditions, we identified the fitness-related functional categories that were affected by light. A set of light-responsive genes involved in osmotic stress tolerance stood out, and a further investigation identified how pre-exposure of Pss B728a to light could enhance fitness upon subsequent exposure to osmotic stress.
Photosensory proteins are used to sense light
Light is an environmental signal that appears daily in the lives of most organisms on the
Earth’s surface and drives several major biological processes, such as photosynthesis, phototaxis, and often development. Photosensory proteins are used to sense light by a variety of organisms.
These organisms are both phototrophic and non-phototrophic. Some fungi and bacteria and all cyanobacteria and plants contain photosensory proteins. Phototrophic and non-phototrophic bacteria make important life decisions with the presence of photosensory proteins (11; 27). A variety of types of photosensory proteins exist; these differ in the wavelengths of light that they 3 sense based on their chromophore. Six families of photosensors have been identified: rhodopsins, phytochromes, xanthopsins, cryptochromes, phototropins, and BLUF (Blue light sensing using
FAD) proteins (79). Whereas this thesis focuses only on the photosensory proteins present in Pss
B728a, namely a blue light sensing protein called the LOV (Light, Oxygen, Voltage) protein and two red/far-red light sensing bacteriophytochromes, the other families of photosensors will be briefly described here. The structure and biochemistry of photosensory proteins has been well studied, but their ecological role in microbes has only recently begun to be investigated.
Diverse photosensory proteins beyond those in Pss B728a
Rhodopsins sense blue and green light and have been identified in eukaryotes, archaea, proteobacteria, cyanobacteria, and vertebrate retina (79). Photosensing in rhodopsins occurs through a conformational cis-trans shift upon adsorption of light by the retinal chromophore.
Rhodopsins have been shown to have roles in flagellar rotation, swimming, phototaxis, and proton pumping. The signal transduction pathways downstream of the rhodopsin photosensors are unknown in many cases (79).
Xanthopsins are blue light-absorbing photosensory proteins with a p-coumaric acid chromophore found in some halophilic bacteria. The most well-known xanthopsin is PYP
(Photoactive Yellow Protein). The sensing of blue light by PYP leads to isomerization within the chromophore like that of rhodopsins. PYP has been shown to have a role in regulating gene expression in some organisms (79).
Cryptochromes have been identified in prokaryotes and eukaryotes, including humans, plants, animals, and algae. Cryptochromes sense blue and green light through a flavin-type chromophore. Photosensing in cryptochromes likely occurs though electron transfer as isomerization of the chromophore is not possible. Cryptochromes have roles in circadian clock regulation, seed germination and seedling elongation, as well as pigment accumulation (79). 4
BLUF proteins have been identified in prokaryotes and eukaryotes. Like cryptochromes,
BLUF proteins also uses a flavin-type chromophore, specifically flavin adenine dinucleotide
(FAD), to respond to blue wavelengths of light. Photosensing in BLUF proteins occurs via electron and proton transfer following excitation of the FAD chromophore. BLUF proteins have been shown to have roles in light avoidance and transcriptional regulation (57; 79).
Photosensory proteins in plants
Phototropins which are LOV domain-containing proteins, cryptochromes, and phytochromes were first discovered in plants. The major role of phototropins is to sense the direction of light and initiate phototropism, which is the bending of the hypocotyl and plant tissue including the stem and petiole towards light for more efficient photosynthesis; this occurs through an auxin gradient (43; 49). Phototropins are also involved in the regulation of chloroplast positioning and stomatal opening. In addition to phototropins, plants contain multiple phytochromes, which are typically referred to as PhyA, PhyB, etc (67). Like LOV proteins, plant phytochromes play a role in auxin accumulation, phototropism, flowering, and growth (20; 28;
43; 49). Similarly to phototropins and phytochromes, plants contain multiple cryptochromes, typically referred to as Cry1, Cry2, etc., which play a role in phototropism, plant growth and development, and circadian clock regulation (50; 79). Phototropin, phytochrome, and cryptochrome pathways intersect and phototropins and phytochromes interact directly to achieve the regulation of plant growth and phototropism (28).
LOV proteins in microorganisms
Pss B728a has one LOV protein and its role in Pss B728a biology is not yet known. LOV proteins sense blue light. LOV proteins have been identified in plants, algae, fungi, and bacteria
(52; 55; 79). Typically, LOV domains consist of an N-terminal sensor next to a linker and output subdomain. The LOV domain binds a flavin cofactor of either flavin mononucleotide (FMN) or 5
FAD. Absorption of blue light by the flavin cofactor causes a transient formation of a covalent bond between the flavin and LOV domain at a conserved cysteine residue, which causes a conformational change in response to the light (32; 35). In the dark, this photoexcited state of the flavin decays back to the noncovalent ground state. Thus, when light is present, the conformational change in the photoreceptor domain is transmitted to an output subdomain downstream of the receptor (27). Approximately 13-15% of sequenced bacterial genomes contain a LOV domain (27; 51). The LOV domain can be found in tandem with many different regulatory domains such as histidine kinases (11), phosphodiesterases, and transcription factors such as sigma factors, sigma factor regulators, and response regulators (32), with approximately
50% of LOV proteins associated with histidine kinase domains.
LOV proteins in bacteria have been shown to influence a range of phenotypes (32). In
Bacillus, LOV proteins affect the general stress response through activation of the stress- associated sigma factor 휎B (32). In Brucella abortus, light exposure leads to a LOV-dependent increase in replication that promotes virulence (32). In Caulobacter crescentus, LOV proteins regulate a blue light-dependent increase in attachment (32). In Synechococcus elongatus, blue light sensing via LOV affects the intracellular c-di-GMP concentration, which leads to regulation of biofilm formation, motility, and virulence (32). In Xanthomonas citri subsp. citri, LOV influences virulence, attachment, biofilm formation, swarming and twitching motility, oxidative stress tolerance, exopolysaccharide production, and flagella production; however, some of these phenotypes are regulated by LOV but independently of light (40). Lastly, in Rhizobium leguminosarum pv. viciae, LOV affects nodulation, flagella production, exopolysaccharide production and attachment (9). The LOV-dependent phenotypes identified thus far are primarily phenotypes that are easily assayed in culture. 6
Phytochromes in microorganisms
The Pss B728a genome encodes two phytochromes. Phytochrome proteins sense red/far red light and typically function as histidine kinases. These proteins have been identified in plants
(68; 77), fungi, cyanobacteria, and a wide variety of bacteria (38; 45; 61). Plant phytochromes bind phytochromobilin at a highly conserved cysteine residue. Phytochromes in cyanobacteria bind phytocyanobilin at the same conserved cysteine (7; 75; 83). Bacteriophytochromes (BphPs) in bacteria (61) bind biliverdin (BV), a product of heme, as their chromophore (8). The formation of linear tetrapyrrole biliverdin occurs when the heme oxygenase acts on the heme ring to open it
(66). In all these phytochromes, the bilin chromophore reversibly interconverts between two stable conformations, a red light-absorbing Pr form and a far-red light-absorbing Pfr form. Like for the rhodopsins and xanthopsins, this response to light is due to a cis-trans isomerization of a double bond in the bilin, with the isomerization mediating a conformational shift in the covalently-bound protein. Non-photosynthetic bacteria may use BV, a precursor to other bilins, as a way of exploiting wavelengths not used by plants or cyanobacteria, since BV responds to longer wavelengths that are not used for photosynthesis (8). Bacteriophytochromes typically contain an N-terminal chromophore-binding domain (6; 17; 81) with conserved PAS, GAF, and
PHY domains (61) that are followed by a C-terminal histidine kinase region (6; 17; 81). In general, histidine kinase domains are predicted to transfer their phosphate to a response regulator that triggers a signal transduction cascade (37), but response regulators for the majority of bacteriophytochromes have not been identified. Bacteriophytochromes are synthesized in a ground state and convert between the ground state and an activated state upon sensing light. In most cases the ground state is the Pr* formation, but some bacteriophytochromes have a Pfr* ground state. These are labeled bathyphytochromes because their absorption maxima are in the bathochromic/long wavelength spectrum regions (72). 7
Bacteriophytochromes in bacteria have been shown to have a wide range of functions. In
Magnetospirillum magneticum AMB-1, the MmBphP1 functions in phototaxis towards red light in an effort to escape oxidative stress, which is a persistent challenge in many environments (15).
Bacteriophytochromes have been identified in greater than 50 purple bacteria, including in
Rhodopseudomonas sphaeroides, Methylobacterium, and Rhodopseudomonas palustris. In most cases, BphP proteins in purple bacteria are responsible for controlling photosynthesis and light harvesting complexes, which can also aid in oxidative stress tolerance (26; 36; 69).
Agrobacterium fabrum contains two bacteriophytochromes, one normal and one bathyphytochrome, and having both of these has been predicted to allow A. fabrum to adapt to a wide range of environmental light conditions (37). However, to date, the only identified role for these AfBphPs is in the light-mediated repression of conjugation (2). The Azospirillum brasilense strain Sp7 contains a bacteriophytochrome that aids in osmotic stress tolerance (42). In
Xanthomonas species, bacteriophytochromes may have a role in downregulating virulence traits to avoid triggering a defensive response within the host plant (3; 10; 74), with a bathyphytochrome in Xanthomonas oryzae pv. oryzae promoting swimming motility, virulence, and iron uptake through siderophore production (80). Collectively, these bacteriophytochromes contribute to a diversity of behaviors that may lead to enhanced fitness upon light sensing.
Photosensory proteins in Pseudomonas syringae
Pseudomonas syringae species are model organisms for studying plant-pathogen interactions. Excitingly, the genomes of most P. syringae strains encode multiple photosensory proteins, namely LOV proteins and bacteriophytochromes, and these proteins are widespread in other plant-associated organisms as well (27; 41). Research on photosensory proteins in P. syringae has primarily focused on the strains Pst DC3000 and Pss B728a. Both strains contain two bacteriophytochromes and a LOV protein. PstLOV plays a role in leaf colonization and 8 virulence, adherence, swarming motility, oxidative stress tolerance, and possibly growth, depending on culture conditions (27; 62; 70; 71). Similarly, PstBphP1 may have a role in epiphytic and apoplastic host colonization, swarming, virulence, and attachment (65; 70; 71; 76).
As similar phenotypes are regulated by both PstLOV and PstBphP1, it is reasonable to hypothesize that they may be interacting in some fashion; however, this has not yet been examined.
In Pss B728a, PssLOV and PssBphP1 have been shown to function in an integrated signaling network where swarming motility, swarm tendril initiation, colonization, lesion formation, and virulence are regulated downstream of light-sensing (58; 59; 84). A few individual genes have been identified that function downstream of PssBphP1 (58; 59); however, the regulatory pathways by which light, PssLOV, and the phytochrome proteins affect these phenotypes are unknown. The lack of clear response receiver proteins for PssLOV and
PssBphP1, and for the homologs in most Pseudomonas spp. genomes, makes identifying the components in these signal transduction systems particularly challenging.
P. syringae can survive in a wide range of environments
P. syringae is commonly thought of as a plant pathogen that is associated with agricultural crops. However, P. syringae has been isolated from a wide range of habitats beyond agricultural ecosystems. These habitats include lakes and streams not exposed to agricultural runoff, snow, rain, natural forests, and prairies (64). Large numbers of P. syringae cells have also been isolated from the air directly above field canopies (47). In line with being airborne, P. syringae cells have been isolated from clouds (1; 19). Taken together, the widespread occurrence of P. syringae across these disparate habitats suggests that this bacterium is highly adaptable. It is important to note that, as this thesis focuses on light sensing in P. syringae, all of the habitats from which P. syringae has been isolated are generally exposed to light. Additionally, P. 9 syringae has been proposed to play a role in the water cycle due to its production of an unusual protein that promotes the formation of ice, called an ice nucleating protein. P. syringae can initiate the formation of ice crystals within clouds leading to precipitation events (54; 60). The movement of P. syringae from leaf canopies, into clouds, and re-dispersal to the ground via snow and rainfall creates a potential lifecycle for these bacteria that is much more complex than as a plant pathogen alone (63; 64).
Pss B728a as a plant pathogen
Pss B728a is a gram-negative bacterium that is pathogenic to the common bean
(Phaseolus vulgaris) as the causal agent of bacterial brown spot. It is known to inhabit a wide geographic range and live as an epiphyte on a large number of host plants (85). Whereas P. syringae is commonly found on heathy tissues, it functions as a pathogen when favorable environmental conditions are met, which include a susceptible host and suitably large populations (33). In general, Pss B728a is well suited to an epiphytic lifestyle (34), as it establishes and maintains populations on non-host as well as host plants without inducing symptoms (16). Moreover, these populations are often on the leaf surface, although as a pathogen it is able to colonize endophytic (or apoplastic) sites (4). Many studies have focused on the ecology of Pss B728a during its growth on and in leaves, with the goal of understanding the genes that contribute to its success in this habitat (29; 30; 46; 56). Here I will touch on just a few of the findings from these studies that highlight some of the traits that make it such a good leaf colonist.
Pss B728a strategies for coping with fluctuating water availability on leaves
Epiphytic Pss B728a populations can be exposed to a multitude of environmental stresses on a daily basis, including, but not limited to, rapid changes in temperature and moisture content
(34), oxidative stress (23), and nutrient limitation (48). The speed at which water availability 10 changes on the leaf surface is a large challenge to epiphytic survival. Whereas a slow loss in water availability allows for accumulation of compatible solutes and secretion of protective polysaccharides, a rapid loss can be lethal. Water loss from leaf surfaces drives an increase in the concentration of leaf surface solutes and can impose an osmotic stress upon the resident bacteria.
If the water loss is rapid, the osmotic upshift can be lethal or at least prevent growth until cellular homeostasis can be restored through compatible solute accumulation. In a study done by Yu et al. (85), a global transcriptome analysis of Pss B728a cells recovered from leaves showed that these cells were exposed to osmotic stress both on leaf surfaces and in the leaf apoplast.
Pss B728a employs several mechanisms to cope with osmotic stress on leaves. One mechanism is through the production and secretion of the exopolysaccharide (EPS) alginate (13;
21; 73; 78). Like other EPS molecules, alginate is hygroscopic and encapsulates the cells, promoting water retention and hydration (18; 39; 82); the expression of alginate synthesis genes in Pss B728a cells on leaves suggests that alginate is produced in the phyllosphere (85). A second mechanism is through the production of syringafactin. Syringafactin is a hygroscopic biosurfactant produced by Pss B728a that increases fitness in the phyllosphere (12).
Syringafactin contributes to local hydration in microsites on leaves by binding tightly to the leaf cuticle and promoting water retention during periods of drying, thus creating a more continuously moist microenvironment despite fluctuations in free surface water (12; 31). A third mechanism is to import plant-derived osmoprotectants and synthesize compatible solutes (44).
Choline is an osmoprotectant that is present on the leaf surface and in the leaf apoplast, likely due to release during the recycling of the plant phospholipid phosphatidylcholine, and this release makes it available for uptake by Pss B728a on the leaves (14). Following uptake, Pss
B728a converts choline to glycine betaine, which accumulates as a compatible solute. Whether 11 choline is present or not, Pss B728a also accumulates compatible solutes by de novo synthesis, and these include N-acetylglutaminylglutamine amide (NAGGN) and trehalose, with NAGGN accumulation generally preceding trehalose accumulation (24; 25; 44).
Goal of the thesis
In this thesis we examine the regulons of several photosensory proteins in the model leaf- associated bacterial strain Pss B728a. Our goal is to better understand the physiological and ecological roles of light sensing in bacterial fitness on leaves. Based on the findings from the regulatory datasets, we examine downstream phenotypes affected by light and photosensory proteins. We place a special focus on responses that increase Pss B728a tolerance to evaporative water loss from leaves, as this is an environmental stress that is temporally linked with light, and thus with photosensing, in the phyllosphere.
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17
CHAPTER 2. A BACTERIOPHYTOCHROME ORCHESTRATES TRANSCRIPTIONAL REPROGRAMMING IN RESPONSE TO MULTIPLE LIGHT WAVELENGTHS IN PSEUDOMONAS SYRINGAE B728a
Bridget Hatfield1, Haili Dong1, Meiling Liu2, Daniel Nettleton2, Xuefeng Zaoh3, Gwyn A. Beattie1.
A modified version of this will be submitted to mBio
1Department of Plant Pathology and Microbiology, Iowa State University, Ames, IA, U.S.A.
2Department of Statistics, Iowa State University, Ames, IA, U.S.A.
3Research IT, Iowa State University, Ames, IA, U.S.A.
All of the data presented in this chapter were generated and analyzed by B.H. with the exception of culture preparation and culture exposure to light prior to RNA preparation and extraction (performed by H.D.), mapping the reads to the genome (performed with the help of
X.Z.), and statistical analysis of the transcriptome data (performed by M.L. and D.N.).
Abstract
An examination of gene expression patterns in response to far-red light, blue light, and red light showed that light is a global regulatory signal in the phyllosphere bacterium
Pseudomonas syringae pv. syringae strain B728a. Collectively, these wavelengths affected the transcript levels of thirty-one percent of the genes, with far-red light influencing the most genes
(1,477) and blue light and red light inducing attenuated responses in fewer genes (1,307 and 675 genes, respectively) but overall similar gene sets. The bacteriophytochrome BphP1 was required for the response of the majority of these genes to each wavelength, and a spectroscopic 18 characterization of purified BphP1 protein confirmed that BphP1 responded to each wavelength.
A blue light-sensing protein with a LOV (light, oxygen, voltage) domain regulated only four genes, and a second bacteriophytochrome, BphP2, did not regulate any genes under the conditions tested. The BphP1 regulon included many osmotic and oxidative stress response genes. The induced, light-dependent genes in the BphP1 regulon significantly overlapped with the induced, osmotic stress-dependent genes in the regulon of the extracytoplasmic sigma factor
AlgU. Together, these findings highlight an integrated response to light and co-occurring environmental stresses in the phyllosphere. Expression of a selected light-responsive gene, algD, increased with light intensity, providing one of the first examples of light intensity-based gene regulation in non-phototrophic bacteria. Light and BphP1 influenced both the expression of two conjugation-related genes and conjugation activity; that is, B728a exhibits phytochrome- mediated, far-red light repression of conjugation. Overall, these data highlight a global response to multiple wavelengths of light, a coordinated response to light and potentially co-occurring osmotic and oxidative stresses in the leaf environment, and a central role for a bacteriophytochrome in this global transcriptional response in P. syringae B728a.
Introduction
Light touches nearly all surfaces on earth, reaching surface waters, soil crusts, and leaf canopies. Many organisms have evolved to utilize light as a signal through light sensing. It is well known that phototrophic organisms like plants, algae, and cyanobacteria exploit light for photosynthesis; however, light-rich environments are rich in non-phototrophic organisms of which some are capable of sensing light. Photosensory proteins are a critical mediator for sensing light in all organisms. Various types of photosensors respond to specific wavelengths of light. These photosensors include BLUF (blue light using flavin adenine dinucleotide) and LOV 19
(light, oxygen, and voltage) proteins and cryptochromes, which sense blue light, and phytochromes, which sense red and far-red light.
LOV and phytochrome proteins are particularly common among plant-associated bacteria
(30; 47), enabling these organisms to respond to a broad spectrum of available wavelengths.
LOV proteins commonly play a role in plant-pathogen interactions by affecting motility, colonization, virulence, and surface adhesion (24; 38; 39; 51; 52; 54; 61; 75). Phytochromes similarly influence plant-pathogen interactions, with phytochromes in two phyllosphere-residing
Pseudomonas syringae strains contributing to colonization, virulence, and swarming (51; 52; 61;
63; 75), and a phytochrome in Xanthomonas campestris regulating virulence by influencing swarming, biofilm production, and the expression of virulence factors (9). Phytochromes regulate additional phenotypes in root-associated bacteria, including conjugation in
Agrobacterium fabrum (2; 42) and oxidative stress tolerance in Azospirillum brasilense (40).
Given the challenge of identifying light-regulated phenotypes through phenotypic assays alone, this list of regulated phenotypes in plant-associated bacteria is likely incomplete. Additional clues to light-regulated phenotypes could be obtained from profiling the effect of light on, and contributions of photosensory proteins to, the transcriptome of these organisms.
A few non-phototrophic bacteria have been examined for the effect of light on their transcriptome. In general, these studies have found that light regulates a large number of genes
(9; 57; 59; 63), although how genetic responses differ across wavelengths has not yet been explored. Two studies have demonstrated roles for phytochromes in gene regulation (3; 8), with one study documenting light-independent phytochrome regulation based on tests performed only in the absence of light (3), and the other showing a role for a phytochrome in far-red light- mediated transcriptome changes (8). At present, few if any studies have comprehensively 20 examined the impact of multiple photosensors and multiple wavelengths on gene expression; such comprehensive studies may provide insights into photosensory protein-specific and wavelength-specific regulons.
Here, we used RNAseq to examine the impact of light and photosensory proteins on gene regulation in P. syringae B728a. Our first objective was to explore how light impacts transcription, with a specific focus on addressing if distinct wavelengths have distinct effects on gene regulation and how this regulation is impacted by the intensity of individual wavelengths.
Our second objective was to investigate if the individual photosensors BphP1 and LOV affect distinct regulons, as they have been shown to work antagonistically in an integrated signaling network in B728a controlling swarming motility, virulence, and colonization (51; 75). We also evaluated if the photosensor BphP2 affects any genes, as BphP2 is not currently known to contribute to any phenotype in B728a. Our third objective was to use the RNAseq results to identify potentially novel light- and photosensor-dependent phenotypes. Collectively these studies are aimed at generating a deeper understanding of photosensor regulons and photosensor- regulated behaviors in the non-phototrophic bacterial species P. syringae.
Materials and Methods
Bacterial strains and growth conditions.
Strains and plasmids used in this study are provided in Table 1. DH5α, a host for cloning, was purchased from Invitrogen and pET21a, a cloning vector for inducible expression of N- terminally T7-tagged proteins, was purchased from Novagen. P. syringae strains were routinely grown in King’s B (KB) medium at 25°C, unless otherwise stated, and Escherichia coli DH5α was grown in Luria (LB) medium at 37°C. Liquid cultures were grown with shaking. Antibiotics were added, when appropriate, as follows: rifampicin (50 µg/mL), chloramphenicol (30 µg/mL), 21 and kanamycin (50 µg/mL). When appropriate, plasmids were introduced into strains by tri- parental conjugation (7; 20).
Table 1. Strains and plasmids used in this study.
Strain or Plasmid Description Reference Strains B728a Wild-type P. syringae pv. (45) syringae; RifR ∆bphOP1 B728a ∆Psyr_3505-3504; RifR (75) ∆lov B728a ∆Psyr_2700; RifR (75) ∆bphOP1∆lov B728a ∆Psyr_3505-3504, (75) ∆Psyr_2700; RifR ∆bphOP1∆bphP2R B728a ∆Psyr_3505-3505, (75) ∆Psyr2385; RifR ∆bsi B728a ∆Psyr_2699; RifR (51) ∆rhlA B728a ∆Psyr_3129; RifR (52) B728a-Cm CmR (This work) BL21(DE3) E. coli cloning host with T7 (68) polymerase gene under the control of Plac, which is IPTG inducible Plasmids pUC18T-miniTn7T mini-Tn7 base vector with (17) transcriptional terminator and oriT pUC18T-miniTn7T-Cm pUC18T-miniTn7 containing (This work) cat inserted as a BamHI fragment, CmR pN pME6041 with nptII promoter (16) adjacent to the multiple cloning site; KmR pRK2073 Conjugal helper plasmid; (7) SmR SpR pPROBE_KT’ Broad-host-range reporter (53) plasmid with ‘gfp; KmR pN-bphOP1 pN containing a constitutively- (75) expressed bphOP1 gene; KmR pN-lov pN containing a constitutively- (75) expressed lov gene; KmR pET21a-bphOP1-His6X bphOP1 operon from B728a (This work) with a C-terminal 6-His tag BphP1 expressed from a Plac- bphOP1 fusion in pET21a 22
Construction of a chloramphenicol resistant B728a strain.
Strain B728a-Cm was constructed by amplifying the cat gene from pHP45Ω-Cm (19) using the primers Cm-F (5’-GCCGGATCCATGGAGAAAAAAATCACTGGATATACC-3’) and Cm-R (5’- TACTGCGATGAGTGGCAGGGCGGGGCGTAAGGATCCGGC-3’), where the nucleotides in bold are BamHI sites. The cat gene was inserted as a BamHI fragment into pUC18T-miniTn7T (17), creating pUC18T-miniTn7T-Cm, and the miniTn7T-Cm was inserted via transposition into the Tn7 insertion site just downstream of the glnS gene.
Construction of pET21a-bphOP1-His6X.
pET21a-bphOP1-His6X was constructed by amplifying the bphOP1 operon from B728a using the forward primer B-OP1-RBS (5’-TCAGGATCCCGGATGCGTTTTCTGCCCATTG-
3’) and the reverse primer B-OP1H6R (5’-TGAGAATTCTCAGTGGTGGTGGTGGTGGTGTC
CGCTTCCGCTTCCAACCGCCATTGGCACCGTG-3’), where the nucleotides in bold are
BamHI and EcoRI restriction sites, respectively. The bphOP1 operon was inserted as a fragment into pET21a (68), creating pET21a-bphOP1-His6X, where 6X His-tagged BphP1 production could be induced with the addition of IPTG.
Lighting conditions.
LED light bars at wavelengths of 730 nm (far-red light), 450 nm (blue light), and 660 nm
(red light) were purchased from BML Horticulture (now Fluence Bioengineering, Austin, TX).
The light bars were equipped with dimming switches that enabled manual control of the intensity, and the light intensities were further controlled by adjusting the height of the light bar.
Wavelengths and intensities were measured using a spectrometer (BLACK-Comet CXR-100,
StellarNet, Inc., Tampa, FL). These conditions provided intensities of 0-10 µmol/m2/sec for far- red light and 0-30 µmol/m2/sec for blue and red light (Fig. S1). White fluorescent growth lights 23 were used at 30 µmol/m2/sec. For all analyses, light-exposed cultures were compared to dark controls, which were obtained by enclosing culture flasks and plates in a double layer of aluminum foil.
Purification of BphP1 protein.
E. coli BL21(DE3) cells containing pET21a-bphOP1-His6X were grown overnight in LB.
Cells were then sub-cultured at 5% volume into a 1L flask and grown at 37°C until an optical density at 600 nm (OD600) of ~0.5 was reached. Culture flasks were then moved to a shaking incubator set to 200 RPM and 17°C and IPTG was added to a final concentration of 1 mM.
Cultures were grown overnight (~18 h) and then centrifuged at ~23,000xg for 10 min. The supernatant was removed and BphP1 was purified using Ni-NTA Agarose (Qiagen) following protocol 9 and protocol 12 in the manufacturer’s instructions (58). Attempts were made throughout the purification process to limit light exposure to retain ground state Pr protein structure.
Spectroscopic evaluation of BphP1 protein.
Purified protein was stored in blackout containers. Aliquots (1 mL) of purified protein were mixed with an excess of biliverdin chromophore to create holoproteins. This solution was loaded into cuvettes and either maintained in the dark or exposed to far-red, blue, or red light for
2.5 h. Immediately after the exposure period, cuvettes were loaded into a spectrophotometer
(Nanodrop 2000) and absorption spectra were measured over 300-820 nm.
Exposure of cells to light for RNAseq analysis.
The strains B728a, ∆bphOP1, ∆bphOP1 (pN-bphOP1), ∆lov, ∆lov (pN-lov),
∆bphOP1∆bphP2R, ∆rhlA, and ∆bsi were examined by RNAseq for their responses to light. Two independent cultures of each strain were grown on each of six separate days. For each culture, an initial culture was grown from a colony in KB medium containing rifampin to a density of appx. 24
109 cells/mL, then 10 µL was sub-cultured into 5 mL KB medium and grown again to a density of appx. 109 cells/mL. A 0.2 mL-aliquot was sub-cultured into 20 mL KB in a 125-mL flask and grown for 4-6 h with shaking in the dark at 20-22°C until mid-log phase, that is, to an OD600 of
0.5. Two mL of the mid-log-phase culture were transferred to a well in each of several 6-well cell culture plates, and the plates were immediately subjected to one of the following conditions for 15 min at 20-22°C without shaking: B728a was exposed to far-red light, blue light, red light, white light, and dark conditions, where dark was imposed by enclosing the microtiter plate in a double layer of aluminum foil; ∆bphOP1 and ∆bphOP1 (pN-bphOP1) were exposed to far-red light, blue light, red light, and dark conditions; ∆lov and ∆lov (pN-lov) were exposed to blue light and dark conditions; and ∆bphOP1∆bphP2R, ∆rhlA and ∆bsi were exposed to far-red light.
Immediately after the dark or light treatment, 0.5 mL of each culture was added to 1 mL of stabilizing reagent (RNAprotect reagent, Qiagen) and vortexed for 5 sec. The cells were incubated in RNAprotect for 5 min at room temperature, and then the cells from the two independent cultures of a strain grown on a single day were combined and centrifuged at 5,000xg for 10 min. The supernatant was decanted, and the cell pellet was stored at -70°C. For each strain, this process was repeated on six separate days. The cell pellets collected on two separate days were combined, and this pool was considered one biological replicate. In this manner, each combination of strain x light condition was represented by three biological replicates, each of which contained cells derived from four independently exposed cultures. In our previous studies we showed that this experimental design supported a robust assessment of the impact of an environmental condition on the P. syringae transcriptome (76).
RNA extraction.
Cell pellets were thawed on ice and the pooled cells collected on two separate days were combined. RNA was extracted using a RNeasy minikit (Qiagen), with one biological replicate 25 for each strain x treatment combination extracted at the same time. DNA was removed using an on-column Dnase I digestion, and RNA quality was evaluated using an Agilent 2100 bioanalyzer. RNA samples (>5μg) were sent to BGI Genomics (Beijing, China) for rRNA depletion, RNA fragmentation, conversion to cDNA, library preparation, and paired-end sequencing using the HiSeq Illumina platform. We obtained an average of 8.8 ± 0.9 million 100- nt reads per sample across the 60 samples, with 98.7 ± 0.1% of the nucleotides with a quality score >20.
Mapping the reads.
The reads for all of the strains were mapped to the B728a reference genome
(GCF_000012245) using TopHat (ver 2.1.0), and the mapped reads were counted by HTSeq- count (ver 0.6.0). Across the samples, an average of 92.3 ± 4.8% of the reads mapped to unique regions and exhibited concordant alignment between the paired-end reads, whereas the remaining reads exhibited alignment to multiple regions or showed discordant alignment.
RNAseq data analysis.
The number of reads per gene were normalized based on the total number of reads per sample by dividing the number of reads per gene by the third quartile of the total read number; this minimized bias from highly expressed genes. The data were then modeled across all treatments for each gene using quasi-likelihood theory which gives an accurate approximation of variance for each gene and therefore a heightened power to determine differentially expressed genes through improved false discovery rate (FDR) estimates (46). The variance term was used in pairwise comparison tests for each gene to test if the transcript abundance differed between treatments. These pairwise comparison tests were performed using the QuasiSeq package in R, where the negative binomial model was used and fixed effects were chosen to be strain x condition (46). FDR values were estimated from p values that were calculated for all pairwise 26 comparisons using the QLSpline method in QuasiSeq. Genes with an estimated False Discovery
Rate < 0.1% (q-value < 0.001) were considered to be differentially expressed.
Characterization of algD expression at various light intensities.
Based on its strong responsiveness to light, the algD gene was selected to evaluate the impact of light intensity on gene expression. Three independent cultures of wild-type B728a were grown in KB medium to an OD600 of 0.6 in foil-enclosed flasks. Aliquots of 2 mL were transferred to 6-well plates and were either maintained in the dark or exposed to various intensities of far-red light (2, 5, and 10 µmol/m2/sec), blue light (2, 10, 20, and 30 µmol/m2/sec), and red light (10, 20, and 30 µmol/m2/sec) for 15 min at 20-22°C without shaking. Immediately after the dark or light treatment, 0.5 mL of each culture was added to 1 mL of RNAprotect stabilizing reagent and RNA was extracted as described above. For each independent culture in each treatment, algD expression was measured in two replicate subsamples using RT-qPCR with a qScript One-Step RT-qPCR Kit (Quantabio, Beverly, MA). The 2-ΔΔCT method (44; 64) was used to calculate the fold-change of each gene in the light treatment compared to in the dark, with rpoD used for normalization.
Conjugation assays.
Tri-parental matings were conducted in KB broth using 6-well cell culture plates. Strains
B728a, ∆bphOP1, ∆lov, and DH5α each containing the plasmid pPROBE-KT’ (53), referred to here as pPROBE, served as donor strains; this plasmid confers kanamycin resistance and is stably maintained in B728a. B728a, ∆bphOP1 and a chloramphenicol-resistant derivative,
B728a-Cm, served as recipient strains. E. coli strain DH5α (pRK2073) was used as a helper strain. Donor, helper, and recipient strains were grown overnight without antibiotic selection.
The cultures were sub-cultured at 10% volume, grown for ~4 h, and adjusted to an OD600 of 1.0, then 100 μL of each strain were transferred to 700 µL of KB broth in wells of the cell culture 27 plates. For all conjugations, at least 3 replicate mating mixtures were prepared, each with a donor culture derived from an independent colony. The cell culture plates with the mating mixtures were exposed to far-red light (10 µmol/m2sec), blue light (20 µmol/m2sec), or dark for 24 h at
20-22°C. After incubation, 100 µL of each mating mixture were plated onto KB agar with kanamycin and rifampin for B728a, or chloramphenicol for B728a-Cm, as recipient. Multiple experiments were performed, with distinct strains and light conditions included in individual experiments.
Statistical analyses.
An over-representation analysis was conducted on the differentially-expressed genes, as described previously, using R and a Fisher’s exact test (77). One-way and two-way Analysis of
Variance (ANOVAs) were performed using the software program JMP Pro 15 (SAS Institute,
Inc) using treatment conditions, strain, and experiment date as factors when appropriate. For the conjugation data, outliers that were identified using Tukey’s box plots were removed from the dataset (62). Student’s t-tests were performed using Microsoft Excel.
Results
Purified BphP1 responds to far-red, blue, and red light in vitro
The responsiveness of BphP1 to distinct wavelengths of light was evaluated using purified BphP1 in vitro. Whole-cell studies suggested that BphP1 from B728a responded to blue light in addition to far-red and red light (75). Here, BphP1 was purified and mixed with biliverdin to form holoproteins, and the holoproteins were either maintained in the dark or exposed to far-red, blue, or red light for 2.5 h. All three wavelengths converted BphP1 from the ground state, which is a red light-absorbing form designated Pr, into a far-red light-absorbing
(Pfr) state (Fig.1), demonstrating that BphP1 responds to blue as well as red and far-red light.
None of the wavelengths promoted a full conversion to the Pfr state. A majority of the protein 28
remained in the Pr state for all wavelengths, with red light converting more protein into the Pfr
state than far-red light, as expected, and far-red light converting more into the Pfr state than blue
light (Fig. 1).
Figure 1. BphP1 responds to far-red, blue, and red light in vitro. Purified BphP1 was combined with biliverdin and maintained in the dark or exposed to far-red, blue, or red light for 2.5 h. Absorption spectra from 300-820nm were measured using a Nanodrop 2000 spectrophotometer. Pr and Pfr peaks are labeled. Pr is the ground state and Pfr is the initial light-converted state for BphP1.
Light is a global signal for B728a gene expression
To evaluate the response of B728a to light, including its responses to distinct
wavelengths of light, we performed RNAseq analysis on B728a cells exposed to far-red, blue,
red, and white light as well as to dark conditions. Cells in KB medium were exposed to far-red
light (730 nm, 10 μmol/m2/sec), blue light (450 nm, 20 μmol/m2/sec), red light (660 nm, 30
μmol/m2/sec), white light (30 μmol/m2/sec), and dark conditions for 15 min prior to isolating
RNA. The distinct intensities were related to the maximum intensities that could be obtained
with our light sources (see Materials and Methods). Genes were identified as exhibiting light-
induced expression changes using a False Discovery Rate (FDR) of < 0.1% (p-value = 0.0011) in
pairwise comparisons between cells incubated in a light condition versus in the dark. Of the
5,220 genes in B728a, only 39 genes had average read counts of <1 across the samples in the 29 experimental dataset; these 39 genes (Table S1) were excluded from the analysis. Of the 5,181 remaining genes, 1,595 genes responded to far-red, red, or blue light (Fig. 2). The measurable impact of light on the transcript abundance of >30% of the genes in B728a indicates that light is a global signal for B728a gene expression. Despite that far-red light was provided at the lowest intensity among the wavelengths examined, far-red light impacted the largest percentage of differentially-expressed genes (93%); this was followed by blue light (82%) and then red light
(42%), with these gene sets significantly overlapping one another (Fig. 2). These gene sets also greatly overlapped with those responding to white light. Of the 1,362 genes regulated by white light, 1,233 also responded to far-red light, 1,147 to blue light, 664 to red light, and 655 to far- red, blue, and red light. The overlap among the gene sets responding to distinct wavelengths is
Figure 2. A large proportion of the genes in P. syringae B728a responded to light, with significant overlap among the genes responding to distinct wavelengths. The numbers in parentheses indicate the total number of light-responsive genes in each treatment. Light- responsive genes, identified as those with a FDR < 0.1% in pairwise comparisons of far-red, red, and blue light treatments with the dark treatment, included genes that were increased and decreased in their transcript levels. White light-responsive genes were not included in the analysis shown. 30 similar when distinct FDR values were used in designating genes as differentially expressed
(Table 2), with most wavelength-specific genes designated as responding to other wavelengths if less stringent FDR values were used (data not shown). These findings indicate a surprising lack of wavelength-specificity in the B728a genes that respond to light; however, they also illustrate a heightened response to far-red light, as illustrated by the ability of more genes responding to far- red light than other wavelengths to cross any single selected significance threshold (Table 2).
Table 2. Differentially regulated genes at a range of FDR values in B728a exposed to light compared to the dark Differentially expressed genes in Differentially expressed genes in multiple wavelengths FDR Far-red Blue Red Far-red and Far-red and Far-red, Blue, Blue Red and Red 0.001% 869 759 356 701 354 351 0.01% 1120 1004 468 915 467 463 0.1%** 1447 1307 675 1194 669 662 1% 2013 1810 1097 1634 1076 1037 **Denotes the FDR value (0.1%) selected to define differentially-expressed genes in this work.
Of the 662 genes that were regulated by far-red, blue, and red light, 413 were upregulated and 249 were downregulated. For most of the 413 genes that were upregulated, far-red light promoted stronger induction than blue or red light, and blue light promoted stronger induction than red light (Fig. 3A). This pattern was similar for the downregulated genes where far-red light promoted stronger repression than blue or red light, and blue light promoted stronger repression than red light (Fig. 3B). Note that the fold change of repressed genes is 10 times less than that of the induced genes. These data further illustrate that gene expression in B728a is more sensitive to far-red light than blue or red light and is more sensitive to blue light than red light. 31
far-red vs red far-red vs blue blue vs red A 30 30 30 Far-red Far-red Blue 25 25 25
20 20 20
15 15 15
Blue
Far-red Far-red 10 10 10
Fold Change Fold 5 5 5 Red Blue Red 0 0 0 0 5 10 15 20 25 30 0 5 10 15 20 25 30 0 5 10 15 20 25 30 Fold ChangeRed Fold BlueChange Fold RedChange
B -1.0 -1.0 -1.0 Red Blue Red -1.5 -1.5 -1.5
-2.0 -2.0 -2.0
Red
Far-red Far-red
-2.5 -2.5 -2.5 Fold Change Fold Far-red Far-red Blue -3.0 -3.0 -3.0 -3.0 -2.5 -2.0 -1.5 -1.0 -3.0 -2.5 -2.0 -1.5 -1.0 -3.0 -2.5 -2.0 -1.5 -1.0 Fold RedChange Fold BlueChange FoldBlue Change
Figure 3. Far-red light had a greater effect than red or blue light on the level of induction (A) and repression (B). The fold-change in expression in light versus the dark is shown for the 413 genes that were induced (A) and the 249 genes that were repressed (B) under far-red, blue, and red light (FDR < 0.1%).
BphP1 regulates the majority of the light-responsive genes in B728a
B728a contains a bacteriophytochrome, BphP1, for which phenotypic assays demonstrate responses to far-red, blue, and red light (51; 75), consistent with the spectroscopic changes in the purified BphP1 holoprotein driven by these wavelengths (Fig 1). The bphP1 gene is in an operon with the bphO gene encoding heme oxygenase, which is responsible for converting heme into the biliverdin chromophore used by BphP1 to sense light. To determine the impact of BphP1 on light-regulated gene expression, a double mutant lacking bphP1 and bphO, called ΔbphOP1, and
ΔbphOP1 expressing the bphOP1 operon on the plasmid pN-bphOP1, were also included in the
RNAseq experiment, with each of these strains exposed to the far-red light, blue light, red light, and dark conditions described above. Of the 1,477 genes responding to far-red light in the wild type, 1,157 (78%) lost this regulation upon deletion of bphOP1 (Fig. 4), indicating that BphP1 regulated the majority of the far-red light-responsive genes. Similarly, we found that BphP1 32 regulated 68% of the blue light- and 74% of red light-responsive genes. Moreover, among the genes induced by these wavelengths, the level of induction in the wild type was strongly correlated with the extent of reduced expression upon the loss of bphOP1 (Fig. S2), indicating that BphP1 is critical to photoregulation of these genes. Expression of bphOP1 in the ΔbphOP1 mutant restored almost all these genes to a wild-type level of expression. The primary exception was Psyr_3503, which showed reduced expression in the ΔbphOP1 mutant indicating that it is a
Figure 4. BphP1 regulates the majority of light-responsive genes in B728a cells exposed to far-red, blue, or red light. Light-responsive genes are represented by the colored bars and BphP1-regulated genes are represented by the grey inset bars. Percentages of the light- responsive genes that are BphP1-regulated are indicated. BphP1-regulated genes were those that were both differentially regulated in B728a under light compared to the dark and in ∆bphOP1 compared to B728a in the indicated light condition. member of the BphP1 regulon but exhibited increased expression in ΔbphOP1 (pN-bphOP1) over B728a under all the conditions tested. This gene, which encodes a hypothetical protein, is directly downstream of bphOP1 but in the reverse orientation. These findings suggest that the expression of this gene is normally attenuated by transcriptional readthrough from the bphOP1 operon. Collectively, these data demonstrate a central role for the bacteriophytochrome BphP1 in the photoregulation of up to 22% of the 5,181 genes examined in B728a, supporting a role for
BphP1 as a global regulator. Moreover, the results illustrate a role for BphP1 in blue light 33 regulation, as demonstrated in Fig. 1, in addition to the red light and far-red light regulation characteristic of phytochromes.
B728a contains a second photosensory protein, LOV, which responds only to blue light and functions with BphP1 in a pathway that affects leaf colonization, lesion formation, and swarming motility by antagonizing the repression of BphP1 (51; 75). We found that the lov gene was induced by far-red, blue, red, and white light in B728a (2.7- to 3.6-fold), and this induction required BphP1. We also investigated the impact of LOV on gene expression by looking for genes that were differentially regulated in a Δlov mutant and Δlov (pN-lov) as compared to the wild type under blue light and in the dark. Surprisingly, we did not find evidence that LOV has a major impact on gene expression; in fact, the Δlov mutant showed reduced transcript levels of only one gene, Psyr_0456. This gene encodes a cytochrome B561. However, lov expression in
Δlov (pN-lov) did not restore Psyr_0456 expression, although it increased lov transcript levels
2.3-fold over those in the wild type. When the stringency was relaxed to an FDR of 1%, two additional genes, Psyr_0457 and Psyr_0715, were identified as differentially expressed, but expression of these also was not restored in Δlov (pN-lov). The lack of differentially expressed genes in Δlov, combined with our previous knowledge of LOV-regulated phenotypes in B728a, suggests that LOV regulation of these phenotypes is mediated at the post-transcriptional level.
Two additional proteins, Bsi and RhlA, function downstream of BphP1 in the photosensory pathway (51). Bsi was identified as an inhibitor of bacteriophytochrome-mediated swarming, whereas RhlA is a synthase of the surfactant 3-(3-hydroxyalkanoyloxy) alkanoate
(HAA). We examined the transcriptome of the ∆bsi and ∆rhlA mutants under far-red light. We confirmed the loss of the bsi transcripts in ∆bsi compared to in the wild type but did not identify other genes that were differentially regulated in this mutant. Bsi, like LOV, may regulate genes 34 at a post-translational level if it is involved in regulation. We did not find evidence for rhlA expression under these conditions, which is consistent with a role for surfactants on surfaces but not necessarily in liquid cultures (10; 77).
We also tested for a role of a second phytochrome, BphP2, and its response regulator,
BphR, on gene expression, although loss of bphP2 has not yet been associated with a known phenotype. To identify potential downstream components of BphP2, we compared the transcriptome of a strain deficient in BphOP1 and BphP2R to a strain deficient in BphOP1 under far-red light. The ∆bphOP1∆bphP2R mutant did not show altered expression of any genes beyond those regulated by BphP1, providing evidence that BphP2 does not influence gene expression in B728a under the conditions tested. Even though we did not find genes that were differentially regulated by LOV, Bsi, RhlA or BphP2, the inclusion of these mutants in our dataset greatly enhanced our statistical power by contributing to better estimates of the variance for each gene.
Over-representation analyses identify functional groups that are strongly regulated by light
Over-representation analyses were conducted to identify the functional categories that contained a disproportionate number of light-responsive genes (identified at an FDR<0.1%) (Fig.
5). Using a Fisher’s exact test, we identified functional categories for which the proportion of induced genes that were differentially expressed in a category were greater than the expected proportion of differentially expressed genes in that category based on the proportion of differentially expressed genes across the genome for each comparison of far-red, red, and blue light to the dark in B728a; a similar analysis was performed for the repressed genes. Among induced genes, several categories related to osmotic stress tolerance were over-represented; osmotic stress tolerance genes will be described in more detail below. Additionally, genes related to extracytoplasmic functions (ECFs), including outer membrane proteins, sigma factors 35
(particularly the ECF sigma factors), transport, and the type 6 secretion system (T6SS) were also over-represented among the induced genes. Among repressed genes, many genes involved in primary metabolism and cellular replication were over-represented. These included genes related to transcription, translation, energy generation, and the metabolism of nucleotides and DNA, lipopolysaccharides (LPS), phospholipids, cofactors, amino acids, and fatty acids.
Figure 5. Over-representation analyses reveal functional groups that have a high proportion of genes that are differentially induced (top) or repressed (bottom) in B728a in far-red, blue, or red light compared to the dark. Over-representation analyses were conducted in R and significance was determined using a Fisher’s exact test on genes that were determined to be differentially expressed based on FDR < 0.1%. (**) represents significance on FDR < 5.0% and (*) represents significance on FDR < 10.0%. Graphs show the percentage of genes regulated in each functional group that was significant under at least one light condition. The graphs on the right show the total number of genes in the genome assigned to the functional category (77).
36
Overexpression analyses were also performed for genes that were differentially regulated in ΔbphOP1 compared to the wild type (WT) under all light conditions. Functional categories identified as disproportionately regulated by BphP1 were similar, though not identical, to those disproportionately regulated by light. One functional category, “proteases”, was not over- represented among the induced genes in ΔbphOP1. Furthermore, induced genes in ΔbphOP1 included new over-represented functional categories, namely “cyclic di-GMP cyclase proteins” and “transport (inorganic ions)”; however, a large number of genes in both of these categories were regulated in response to light in the WT, despite the category not being identified as over- represented. Among the repressed genes, the ΔbphOP1 response did not have an over- representation of genes in the categories of “fatty acid metabolism”, “oxidative stress tolerance”,
“phospholipid metabolism”, “post-translational modification”, or “siderophore synthesis and transport”. However, there was one additional repressed functional category in ΔbphOP1,
“carbohydrate metabolism and transport”. Collectively, these data suggest that light prompted a change in the pathways used for primary metabolism and that BphP1 contributed to most, but not all, of the light-induced transcriptional changes in functional categories associated with a metabolic shift.
Interestingly, some genes were regulated by a specific wavelength of light. In particular, blue light increased expression of more oxidative stress tolerance genes than did red or far-red light (Fig. 5). While a large number of oxidative stress tolerance genes were regulated under all wavelengths of light tested, blue and white light greatly increased the magnitude of the induction for five oxidative stress tolerance-related genes compared to the dark (Fig. 6). BphP1 had little or no role in this induction, as this regulation was not altered in the ΔbphOP1 mutant for most of these genes. The primary exceptions were ahpF and ahpC, for which their expression in 37
ΔbphOP1 differed slightly, but significantly, from their expression in B728a under blue light; a similar comparison could not be made in white light since we did not evaluate the transcriptome of ΔbphOP1 under white light. The lack of a major role for BphP1 in blue light-mediated oxidative stress gene induction is supported by the apparently small, light-specific role of BphP1 here as compared to for most genes where BphP1 regulation is large in magnitude and measurable under all light conditions. This BphP1-independent, blue light-induction of oxidative stress tolerance genes may reflect a toxicity of blue light as a higher energy wavelength than red or far-red light (26; 69).
Figure 6. Genes involved in oxidative stress tolerance were induced by blue and white light. The fold-change values are shown for three catalase-encoding genes (katA, katB, katG) and two alklhydroperoxidase-encoding genes (ahpC and ahpF) under blue, white, red, and far- Genesred light involved compared in osmotolerance to the dark. Significance are prominent is based among on an light FDR- andof 0.1%. BphP1 -induced genes
The over-representation analyses above highlighted that genes involved in osmotic stress tolerance were disproportionately induced by light and by BphP1 under each light condition examined (Fig. 5, data not shown). The genes within functional categories related to osmotic stress tolerance were among those most strongly induced by light (Fig. 7). These categories included the synthesis and metabolism of quaternary ammonium compounds (QACs), 38 polysaccharide synthesis and regulation, mechanosensitive ion channels (MSCs), and compatible solute synthesis. QACs include the strongly osmoprotective betaines and their precursor choline.
The genes that were induced are primarily involved in the uptake of these osmoprotectants (13;
14; 16) (Fig. 7).
Figure 7. Genes involved in water stress tolerance were strongly induced by each of the light conditions. The fold-change values under light compared to dark conditions are shown for B728a genes representing distinct functional categories influencing osmotolerance. These genes were also regulated by BphP1 based on that induction was lost in ΔbphOP1 under each light condition (data not shown). Gene numbers are as follows, from top to bottom: Psyr_4709, 4827, 4249, 0063, 1063, 1057, 4446, 0040, 4477 4276, 2993, 2490, and 3747.
The polysaccharide synthesis and regulation category included genes involved in the synthesis and regulation of alginate, which is known to aid B728a in osmotolerance (22), and glucan, which is has been shown to be an important factor in response to desiccation stress in P. aeruginosa (73). A total of 54% of genes in the polysaccharide synthesis and regulation category were induced under far-red light, with far-red light inducing 22 of 26 genes involved in alginate synthesis and regulation and all three glucan synthesis genes. Furthermore, 85% of the light- regulated polysaccharide synthesis and regulation genes were regulated by BphP1. 39
Four of the seven genes in the MSC category were induced by light and were regulated by BphP1. MSCs are crucial for responding to hypo-osmotic stress in bacteria by sensing a change in membrane turgor and facilitating a release of solutes and osmolytes. While induction of MSC genes would only be expected to occur in situations with an excess of water, previous data has shown that MSC genes are upregulated in the presence of NaCl in B728a (76).
Additional work needs to be done to investigate if MSC could be involved in the import of osmolytes as well as their export.
Another functional category containing genes involved in osmotolerance is the compatible solute synthesis group. This group is comprised mainly of genes coding for trehalose and N-acetylglutaminylglutamine amide (NAGGN), which are two major compatible solutes synthesized by B728a (21; 22). This category also contains one gene, ectC, for ectoine synthesis, another compatible solute; however, B728a lacks ectA and ectB, genes which are required for ectoine synthesis (41). Whereas ectC is not regulated by light, the majority of the NAGGN and trehalose synthesis genes were upregulated under all light conditions in B728a. Previously we reported that B728a accumulates glutamate as an additional compatible solute through downregulation of the glutamine synthetase gene glnA1 under osmotic stress conditions (22).
Interestingly, the current data also show a downregulation of glnA1 under all light conditions, consistent with glutamate accumulation in response to osmotic stress. Additionally, 9 out of 12 predicted hydrophilins (22), which are small hydrophobic proteins capable of promoting water limitation tolerance (23; 60), were upregulated in B728a in response to light. These patterns of induction were lost in the ∆bphOP1 strain. Taken together the light- and BphP1-regulation of genes in functional groups related to osmotic stress tolerance mirrors that of cells experiencing a water stress, suggesting integration of these environmental signal-sensing pathways. 40
The light-dependent BphP1 regulon overlaps with the osmotic stress-dependent AlgU regulon
AlgU is an ECF sigma factor that regulates the expression of genes involved in osmotic and oxidative stress responses (36; 37; 49), including genes involved in regulating the production of alginate (48). We previously characterized the AlgU regulon in B728a (22). A comparison of the AlgU regulon genes induced under hyperosmotic conditions and the BphP1 regulon genes induced under far-red light conditions showed an overlap of 407 genes (Fig. 8). This intersection contained 72% of the BphP1-induced genes and 85% of the AlgU-induced genes. This large overlap between the two regulons further supports that the responses to light and osmotic stress are integrated in B728a.
Figure 8. Far-red light-induced genes in the BphP1 regulon overlap with osmo-induced genes in the AlgU regulon. AlgU-regulated genes (76) were selected based on FDR <1.0% and BphP1-regulated genes were selected based on FDR < 0.1%.
AlgU-independent, BphP1-regulated genes included eftA, a gene required for epiphytic fitness under dry conditions (5; 6), three genes involved in the heat stress response including the sigma factor σH (rpoH, dsbA, and hsp70), genes involved in glutathione metabolism (pepN, gor, and Psyr_2499), and genes predicted to contribute to the evasion of host defenses (effectors,
PAMPs). These data support a role for BphP1 in regulating traits related to fitness in the 41
phyllosphere in an AlgU-independent manner, in addition to its involvement in environmental
stress tolerance in an AlgU-dependent manner, as described above.
BphP1 disproportionately induces genes that encode membrane-localized proteins
BphP1 is predicted to localize to the cytoplasmic membrane (74), similar to the
regulatory proteins that interact with AlgU (28), and to regulate the expression of ECF sigma
factors and proteins that have an extracytoplasmic location (Fig. 5). Given these findings, we
investigated if there were trends in the predicted localization of proteins within the BphP1
regulon. We performed an over-representation analysis of BphP1-regulated genes based on
protein localization using Fisher’s exact test. Many genes encoding cytoplasmic membrane
proteins and unknown proteins were induced (Fig. 9A) and few were repressed (Fig. 9C). In
contrast, few genes encoding cytoplasmic proteins were induced (Fig. 9B) and many were
Figure 9. BphP1 disproportionally regulates genes encoding predicted membrane- associated proteins. Over-representation analysis using Fisher’s exact test was performed for genes induced (A, B) or repressed (C, D) under far-red, blue, and red light based on predicted protein localization sites. (**) represents significance on FDR < 5.0% and (*) represents significance on FDR < 10.0%. 42 repressed (Fig 9D). The disproportionate upregulation of genes encoding membrane-localized proteins and downregulation of cytoplasmic proteins highlights the modulation of cell-envelope stress as one potential function of the BphP1 regulon. algD expression is sensitive to both quality and quantity of light
To evaluate the effect of light intensity on gene expression in B728a, we identified a strongly light-responsive gene that could be used as an indicator gene. We quantified the expression of the EPS synthesis gene algD using RT-qPCR with RNA extracted from B728a cells that had been exposed for 30 min to a range of intensities for each light wavelength, namely to far-red (2-10 µmol/m2/sec), blue (2-30 µmol/m2/sec), or red (10-30 µmol/m2/sec) light. algD increased in expression with intensity under each far-red, blue, and red light (Fig. 10). At low intensities (≤10 µmol/m2/sec), the magnitude of algD upregulation was much greater under far- red light than under blue or red light, consistent with our previous conclusion that B728a exhibits much greater sensitivity to far-red light than to the other wavelengths (Fig. 3). Only blue and red light could be tested at higher intensities due to our experimental setup. At these intensities (>20
µmol/m2/sec), algD was induced more under blue light than red light, consistent with our previous conclusion (Fig. 3). The similarity in blue light induction at 20 and 30 µmol/m2/sec indicates that 20 µmol/m2/sec was sufficient to provide maximal induction and that an upper light limit exists for induction, at least for blue light. 43
Figure 10. algD expression increases in response to increasing intensities of blue, far-red, and red light. RT-qPCR was used to quantify algD expression using RNA extracted from B728a cells exposed for 30 min to the indicated intensities of blue light, far-red light, red light, and white light. The fold-change in expression in the light compared to in the dark (2-∆∆CT values) is shown. Significance was determined using a one-way ANOVA.
BphP1 regulates conjugation in a light and wavelength specific manner
We used the mapping function of the KEGG database (33-35) to map the BphP1-
regulated genes to cellular pathways and functions. We identified two BphP1-regulated genes,
Psyr_4643 and Psyr_5134, that were predicted to influence conjugation. A study by Lamparter
and colleagues (2) has shown that the bacteriophytochromes Agp1 and Agp2 regulate
conjugation in Agrobacterium fabrum, indicating precedence for phytochrome control of
conjugation. Psyr_4643 is annotated as a conjugal transfer protein (74). This protein has an
ortholog, Trb, in Rhizobium leguminosarum bv. viciae that induces plasmid transfer (18; 43; 50).
Psyr_4643 expression was downregulated in B728a and upregulated in ∆bphOP1 in response to
far-red light as compared to the dark (Table 3) and did not change significantly in blue or red 44 light in either strain. These data provide evidence that BphP1 negatively regulates Psyr_4643 under far-red light.
The other BphP1-regulated gene predicted to be involved in conjugation, Psyr_5134, is annotated as an inner membrane translocase component (74) and has an ortholog in
Enterococcus faecalis, CcfA. CcfA is a precursor for the peptide pheromone cCF10, which is a conjugation signal (1). Our data show that Psyr_5134 was upregulated in ∆bphOP1 as compared to in B728a under far-red and blue light but did not change significantly under all wavelengths examined when wild type B728a was compared to the dark (Table 3). These data suggest that
BphP1 also negatively regulates Psyr_5134 under far-red, as well as blue, light conditions.
Table 3. BphP1 regulates two putative conjugal transfer genes.
Fold-change in B728a in the light as Fold-change in ∆bphOP1 as compared compared to in the darka to in B728aa Gene Far-red Blue Red Far-red Blue Red Psyr_4643 -1.5* -1.2 -1.2 1.3* 1 1.1 Psyr_5134 -1.3 -1.3 -1.2 1.2* 1.2* 1.2 a Fold-changes that were significant at an FDR <0.1% are indicated with *.
Based on BphP1- and light-mediated downregulation of Psyr_4643 and Psyr_5134 and the contribution of their orthologs Rhizobium leguminosarum Trb and Enterococcus faecalis
CcfA, respectively, to conjugation, we hypothesized that light and BphP1 repress conjugation in
B728a. We tested this hypothesis using the mobilizable plasmid pPROBE, which had been introduced into B728a, as the donor and a chloroamphenicol-resistant derivative of B728a,
B728a-Cm, as a recipient in conjugations conducted in the presence of an E. coli strain expressing the helper plasmid, pRK2073, to mobilize pPROBE. We performed these tri-parental matings in liquid culture under far-red, blue, and dark conditions for 24 h at the light intensities used in the RNAseq study, quantifying conjugation based on the number of transconjugants that 45 were generated after introducing the same density of each parent in all mating mixture. Mating mixtures exposed to far-red light had fewer transconjugants than mixtures kept in the dark (Fig.
11), indicating that far-red light decreased conjugation in B728a. Blue light did not decrease conjugation, and maybe even increased it (Fig. 11).
Figure 11. Far-red light, but not blue light, reduced conjugation in B728a. Mating mixtures of B728a (pPROBE), B728a-Cm (pPROBE), and DH5α (pRK2073) were placed under far-red or blue light conditions or dark conditions for 24 h and then plated to enumerate transconjugants. For each wavelength, conjugation data from two independent experiments were combined and analyzed in a two-way ANOVA where the factors were experimental replicate and treatment, with each experimental replicate having n = 10 (far-red light) or n = 12 (blue light). The p-values for the treatment effect in the two ANOVAs were 0.04 (far-red light) and 0.07 (blue light). Next, we evaluated the effect of the photosensory proteins BphP1 and LOV on conjugation under far-red or blue light. We compared the efficiency with which ∆bphOP1 and
B728a served as donors for the plasmid pPROBE to B728a-Cm as a recipient under far-red light.
∆bphOP1 supported the generation of more transconjugants than B728a (Fig. 12), indicating that
BphP1 functions in the donor as a negative regulator of conjugation under far-red light. In contrast, no differences were found between B728a (pPROBE), ∆bphOP1(pPROBE), ∆lov
(pPROBE), or ∆lov∆bphOP1 (pPROBE) versus B728a in their ability to transfer the pPROBE 46 plasmid into the B728a-Cm recipient strain under blue light (Fig. 13), indicating that these photosensory proteins did not play a role in conjugation under blue light.
Figure 12. The bacteriophytochrome BphP1 is a negative regulator of conjugation under far-red light in B728a. Mating and analysis were conducted as in Fig. 11. Graph is of three replicate experiments with n = 11 in each replicate experiment, analyzed in a two-way ANOVA, with the treatment effect showing a p-value of 0.005
Figure 13. BphP1 and LOV do not affect conjugation under blue light. B728a (pPROBE), Δlov (pPROBE), ΔbphOP1 (pPROBE), and ΔbphOP1Δlov (pPROBE) were used as donor strains. Mating and analysis were conducted as in Fig. 11. Data represent three replicate experiments, each with n = 12, and were analyzed using two-way ANOVA, with the treatment effect showing a p-value of 0.51. . 47
We also tested the effect of BphP1 on plasmid uptake by B728a and ΔbphOP1 under far- red or blue light. The donor strain used was E. coli DH5α (pPROBE) and the helper strain was E. coli DH5α (pRK2073). BphP1 did not affect plasmid uptake under far-red or blue light (Fig. 14), indicating that BphP1 does not have a role in the recipient strain. Taken together, these data reveal conjugation as a novel light- and BphP1-regulated phenotype in P. syringae B728a, where far-red light negatively regulates plasmid donation via BphP1.
Figure 14. BphP1 does not affect plasmid uptake under far-red light (A) or blue light (B). Conjugation was conducted as in Fig. 11, with n = 12 for B728a (pPROBE) and n = 11 for ΔbphOP1 (pPROBE). A Student’s t-test was performed to determine significance between the strains, where p = 0.58 (A) or 0.17 (B).
Discussion
Here we present a factorial study characterizing the transcriptional responses of wild-type and photosensory protein-deficient mutants of B728a to multiple wavelengths of light; this study represents the most comprehensive look, to date, at the non-phototrophic bacterial response to light. In addition to finding that light affects the expression of approximately one-third of the
B728a genes, we discovered that B728a exhibits a heightened response to far-red light as compared to blue and red light, but that the gene content of the transcriptional response exhibits 48 little wavelength specificity. Moreover, the responses to far-red, blue, and red light depend on the bacteriophytochrome BphP1 with little to no detectable involvement of second bacteriophytochrome, BphP2, or a blue light-sensing LOV protein. This far-red light sensitivity may reflect an amplification of far-red light within foliar plant tissues due to a lack of far-red absorption by plant chlorophyll, and a potential evolutionary adaptation of this leaf resident bacterium to the environmental signal within leaves.
Our results complement the few previous studies exploring the effect of light and bacteriophytochromes on transcription in non-phototrophic bacteria. In general, these studies similarly found that light influences the expression of a large number of genes. For instance, low-intensity broad-spectrum light induced global transcriptome reprofiling in Listeria monocytogenes (57), and green light affected the expression of 243 genes in Pseudomonas cichorii (59). Similarly, white, blue, and red light induced genetic reprogramming in P. syringae pv. tomato strain DC3000 (63). Two other studies demonstrated a critical role for a bacteriophytochrome in gene expression, including in P. aeruginosa in which 74 phytochrome- regulated genes were identified although the regulatory dependence on light was not addressed
(3), and in X. campestris in which nearly 26% of the genome was regulated by light in a bacteriophytochrome-dependent manner (9).
Genes involved in osmotic stress tolerance were among the genes most strongly induced by light. Using over-representation analysis, we found that light and BphP1 disproportionately influence genes in several functional categories related to osmotic stress tolerance. These categories include genes involved in the synthesis of compatible solutes, polysaccharides, and transporters that can take up osmoprotective compounds, such as choline, which is abundant and available to B728a in plant leaves (15). Our finding is consistent with previous reports that light 49 upregulates osmotic stress tolerance genes in P. aeruginosa (3) and P. syringae DC3000 (3; 63).
Our data indicated that one of the genes regulated by light and BphP1 is algU, which encodes an
ECF sigma factor. Previously, our lab characterized the AlgU regulon in B728a, showing that it regulates >800 genes in the presence of osmotic stress, but <30 genes in the presence of stresses that do not impose cell envelope stress, including oxidative stress and nitrogen and iron starvation (76). Comparison of the induced genes in the BphP1 regulon under far-red light to those in the AlgU regulon under osmotic stress revealed an overlap in the majority of genes in the two regulons, with 85% of the induced AlgU-regulated genes falling within the BphP1 regulon. These data highlight a coordinated biological response to light and osmotic stress.
Osmotic stress occurs on leaves whenever there is a period of surface drying due to the increase in concentration of surface solutes. Given that moisture collects as dew on leaves in the night, and this dew generally evaporates in the morning hours, this coordinated biological response to light and osmotic stress may reflect adaptation to co-occurring environmental signals, including the increase in light, temperature, and drying that follows dawn.
Most light-responsive genes were regulated in a BphP1-dependent manner in response to all three wavelengths tested, including several genes involved in the response to oxidative stress.
However, a subset of oxidative stress tolerance genes was upregulated in blue light only and in a
BphP1-independent manner. Given that the blue light-sensing protein, LOV, regulated very few genes at the transcriptional level in this work, the identity of the sensor for this regulation is unclear. Several studies have shown that blue light induces the formation of reactive oxygen species (ROS) in bacteria (56; 70), and these ROS are harmful to proteins, membranes, and other cellular building blocks (12; 27; 29). White and blue light upregulate oxidative stress tolerance genes in P. syringae DC3000 (63), and Santamaria-Hernando et al. postulate that light, water 50 limitation, and oxidative stresses might all peak at mid-day, leading to a co-association of their response pathways in DC3000 (63). Here, we cannot differentiate between the possibility that blue light induces ROS formation in B728a, with the resulting oxidative stress leading to activation of oxidative stress tolerance genes, versus that a blue light-responsive sensor directly induces these genes, perhaps in anticipation of upcoming oxidative stress.
Based on the predicted protein localization of light-responsive genes, we identified a bias towards regulation of genes that code for membrane-associated proteins. While the reason for this regulation pattern is unclear, several mechanisms may come into play regarding membrane protein regulation under light. Light plays a role in the regulation of stress response pathways, and many stresses, including oxidative and osmotic stresses, can affect membranes, including membrane integrity. Regulation of genes that code for membrane-localizing proteins suggests that integration of the response pathways for these three co-occurring environmental signals
(light, osmotic stress, and oxidative stress) may occur through the interaction of regulatory proteins in the membrane or extracytoplasmic spaces. Envelope stress responses commonly involve regulating the stability of ECF sigma factors, such as AlgU, through sequestration and modulation by anti-sigma factors and their antagonists, such as MucA, MucB and AlgW (72).
Here, we showed that the BphP1 regulon includes several ECF sigma factors, each of which may interact with other membrane-associated proteins to impact gene expression.
Previous work has shown that BphP1 plays a role in swarming, virulence, and colonization in B728a (51; 75). A major goal in conducting the RNAseq experiment was to identify additional phenotypes controlled by light and BphP1. Through mapping BphP1- regulated genes in the KEGG database, we identified light-responsive genes that were related to conjugation. These genes responded to the three wavelengths tested but, similar to most BphP1- 51 regulated genes, exhibited the greatest response to far-red light. Previous studies have shown that bacteriophytochromes affect conjugation in Agrobacterium fabrum by repressing plasmid transfer from donor cells and aiding in plasmid transfer in receiver cells (2; 42). Here, we found that BphP1 in B728a similarly functions as a negative regulator of plasmid donation, and this occurs only under far-red light. However, BphP1 did not detectably affect plasmid uptake into receiver cells. Lamparter and colleagues have suggested that repression of conjugation by a bacteriophytochrome is a mechanism to protect single-stranded DNA from UV light damage (2;
42). Typically, if one strand of DNA is damaged, the second strand is used as a template for repair. During conjugation, only one strand is transferred, so any light-induced damage would be irreparable without the second strand. While this theory is plausible, our data showed that the
BphP1-repression of conjugation occurs under far-red light, which is a low energy wavelength that should cause little to no damage to DNA. Thus, an alternative theory is that conjugation imposes a fitness disadvantage if it occurs when cellular energy is needed for other processes.
For example, light induces genes involved in osmotic and oxidative stress responses, and these responses can be metabolically expensive (31; 32; 65; 71). Therefore, B728a may downregulate conjugation under light so that it can better allocate resources to cope with environmental stresses that co-occur with light.
The finding that far-red light evoked a stronger response than blue or red light despite that far-red light was applied at a lower intensity led us to question how light intensity affects gene expression. The effect of light intensity is well studied in purple phototrophic bacteria where light intensity plays a role in the number of photosynthetic processes (55). Light intensity influences the expression of photosynthetic pigments in phototrophs, as illustrated by blue light intensity effects on the expression of the PUC and PUF light-harvesting complex operons in 52
Rhodobacter sphaeroides (66; 67). This intensity effect is also illustrated by the sensitivity of genes for photosynthetic complexes to low light intensities, as this allows some phototrophs to increase the number of photosynthetic complexes formed under low light (4; 25). The effect of light intensity on non-phototrophic bacteria is not well studied. We explored this by measuring the expression of algD, a differentially expressed gene under far-red, blue, and red light, under increasing intensities of light. We observed a continuous response of algD expression to increasing light intensities rather than a binary response, although in blue light, at least, we observed an upper threshold of activity. The ability to respond in a continuous manner to increasing light intensities, at least in a low range of intensities, may benefit leaf-colonizing bacteria by allowing them to gradually adjust to the environmental changes that accompany the rising sun.
Although BphP1 is required for the regulation of most light-responsive genes, some light- responsive genes were outside the BphP1 regulon; these BphP1-independent genes did not fall into any clear functional categories. We also identified one gene that was BphP1-dependent but independent of light. This gene, Psyr_3503, was positively regulated by BphP1 and codes for a hypothetical protein. Interestingly this gene is located directly upstream of, and divergent to, the bphOP1 operon (Psyr_3504-3505). The ∆bphOP1 strain contains a clean deletion that was confirmed by sequencing; therefore, the change in Psyr_3503 expression was not a result of inadvertent deletion of non-target nucleotides. The divergent orientation to the bphOP1 operon may be related to its altered expression, but this would need to be explored more.
The experimental design employed in this study enabled rigorous evaluation of genes as differentially expressed. In particular, we looked at 22 distinct strain x condition combinations, with three replicates of each, yielding a total of 66 comparisons. Moreover, strains harboring 53 deletions in several genes did not alter gene expression. The deleted genes with little to no transcriptional effect included the photosensory protein genes lov and bphP2, the regulatory gene bsi that functions downstream in the BphP1 regulatory pathway (51), and rhlA, a gene encoding a surfactant (11) that likely is expressed only on solid media rather than in the liquid medium used here. The inclusion in our experimental design of these mutants, which essentially recapitulated the wild type, increased our statistical power to identify differentially expressed genes, allowing us to use a rigorous FDR threshold (< 0.1%). The use of this rigorous threshold, however, may have resulted in missing genes that were differentially regulated, such as additional BphP1-dependent, light-independent genes or light-dependent, BphP1-independent genes. However, the finding that 31% of the B728a genes were regulated by light highlights the importance of light as an environmental cue, since in comparison to cues like osmotic stress, oxidative stress, and heat, visible light generally does not induce stress and thus would not be predicted to induce a transcriptional response as large as a stressor would.
Given the role of light as a global signal for transcription in B728a, we have thus far identified only a few of the phenotypes that light regulates in this organism. Our work expands the demonstrated light-regulated phenotypes in B728a from swarming motility, leaf colonization and virulence as a foliar pathogen to include plasmid donation during conjugation. More importantly, our work at the transcriptional level identifies a range of traits from environmental stress tolerance to the type VI secretion system that are probably impacted by light, and thus provides a foundation for further experimentation probing the physiological benefits of light- sensing by P. syringae and other non-phototrophic bacteria.
Acknowledgements
We thank Dr. Li Ling for her invaluable help in coordinating the sequencing of our samples and mapping of the reads, Dr. Breah LaSarre for her contributions to the editing of this 54 manuscript, Dr. Liang Wu for the construction of B728a-Cm, and Sterling Wells for her help in the optimization and purification of BphP1. This project was supported by the Agriculture and
Food Research Initiative competitive grants program, Award numbers 2010-65108-20562 and
2015-67013-23005 from the USDA National Institute of Food and Agriculture.
References
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Supplementary Data
Supplementary Table 1. 39 genes were excluded from RNAseq analysis due to the average read count across all samples being less than 1.
Gene Number 1 Psyr_0311 2 Psyr_0730 3 Psyr_0732 4 Psyr_0987 5 Psyr_1201 6 Psyr_1449 7 Psyr_1680 8 Psyr_2046 9 Psyr_2200 10 Psyr_2318 11 Psyr_2344 12 Psyr_2548 13 Psyr_2550 14 Psyr_2694 15 Psyr_2765 16 Psyr_2781 17 Psyr_2872 18 Psyr_3379 19 Psyr_3390 20 Psyr_3399 21 Psyr_4728 22 Psyr_5108 23 Psyr_RNA1 24 Psyr_RNA13 25 Psyr_RNA14 26 Psyr_RNA15 27 Psyr_RNA26 28 Psyr_RNA4 29 Psyr_RNA49 30 Psyr_RNA5 31 Psyr_RNA50 32 Psyr_RNA51 33 Psyr_RNA6 34 Psyr_RNA7 35 Psyr_RNA8 36 Psyr_RNA72 37 Psyr_RNA73 60
Supplementary Table 1 Continued Gene Number 38 Psyr_RNA78 39 Psyr_RNA79
Supplementary figure 1. Spectrum of LED light bars centered on (A) 730 nm for far-red light, (B) 650 nm for red light, and (C) 450 nm for blue light. 61
Supplementary figure 2. (A) BphP1 is responsible for the majority of the induced genes under far-red, blue, and red light but (B) BphP1 is not clearly involved in the repression of genes by these wavelengths.
62
CHAPTER 3. PLANT-ASSOCIATED PSEUDOMONADS USE LIGHT AS AN ANTICIPATORY CUE TO ENHANCE OSMOTOLERANCE
Bridget M. Hatfield1, Breah LaSarre1, and Gwyn A. Beattie1
A modified version of this will be submitted to Nature Communications
1Department of Plant Pathology and Microbiology, Iowa State University, Ames, IA, U.S.A
All of the data presented in this chapter were generated and analyzed by B.H. with the exception of the surface water evaporation data, which was generated with the help of B.L.
Abstract
Microbes may respond directly to environmental fluctuations, but also may anticipate future conditions by exploiting re-occurring temporal linkages among these fluctuations. Here, we expand existing knowledge of microbial anticipation by demonstrating that light signals stimulate pre-adaptation of bacteria to subsequent osmotic stress exposure. We demonstrate this anticipatory response in Pseudomonas syringae pv. syringae (Pss) B728a, an organism that is adapted to living on terrestrial plant leaves where diel fluctuations occur in light, temperature, and surface drying. Pss B728a exhibited enhanced osmotolerance after pre-exposure to either red or far-red light, and the response required the red/far-red-light-sensing bacteriophytochrome
BphP1. This response did not require other genes in BphP1-regulated pathways, indicating that light-enhanced osmotolerance represents a new branch of an integrated BphP1-mediated, light- response network. Osmoprotectant transport was essential for light-enhanced osmotolerance under the conditions tested. Measurements of the fluctuations in light and moisture on exposed 63 surfaces on a September day in Iowa demonstrated that light onset at dawn preceded drying of dew-moistened surfaces. These measurements illustrated a temporal coupling that provides a compelling biological rationale for the regulation of osmotolerance by light. We further demonstrated that light-enhanced osmotolerance is conserved in other P. syringae strains and
Pseudomonas species adapted to terrestrial habitats, including P. putida, P. cichorii, and P. protegens. Taken together, this work establishes that bacteria can use light as a predictive cue to mount anticipatory responses against ensuing environmental stresses, and that light-enhanced osmotolerance is a conserved trait among some leaf and root-associated bacteria.
Introduction
A key feature underlying microbial fitness is the ability to sense and quickly respond to environmental changes, particularly those that pose a danger to the cell. Common abiotic challenges to microbial fitness include heat stress, oxidative stress, pH stress, osmotic stress, and nutrient limitation. Stress-coping strategies are most commonly activated by direct exposure to a stress, wherein a cell responds by altering gene expression and cellular activities to minimize cell damage and safeguard essential cell functions. For example, exposure of bacteria to an osmotic upshock triggers the uptake of exogenous osmoprotective compounds and the de novo synthesis of compatible solutes to enable bacterial homeostasis while the environment remains hyperosmotic. These direct responses to stress are often highly coordinated, as illustrated by the activation of alternative sigma factors that coordinately regulate many genes in bacteria. For example, exposure of Pseudomonas spp. to osmotic stress activates the extracytoplasmic function (ECF) sigma factor AlgU, which induces genes for osmoprotectant transport and the synthesis of compatible solutes and the hygroscopic exopolysaccharide alginate, which facilitates hydration (15; 75) 64
Organisms can also employ more complex strategies wherein they use one signal to pre- emptively prepare for subsequent environmental changes. For example, the response to a low stress level may bestow improved tolerance to a high stress level that is encountered later; this protective response has been described as “priming”, “acquired stress tolerance”, and “cross- protection” (25; 56). Cross-protection is also the term used when exposure to one stress signal increases tolerance to an unrelated stress; this may occur via the activation of a general stress response pathway or the simultaneous activation of multiple, sometimes overlapping, response pathways by a single stress, such as the stimulation of the sigma factor RpoS regulon by nutrient starvation, oxidative stress, extreme pH, or osmotic stress. (55). A more specific type of response has been termed “adaptive prediction” (46), wherein an organism exploits the temporal coupling of environmental changes within a habitat to enable “prediction” of upcoming opportunities and threats. For example, upon ingestion by a host, Escherichia coli uses an increase in temperature to anticipate and prepare for an imminent decrease in oxygen availability in the stomach (68). E. coli also uses the presence of lactose to anticipate the subsequent availability of maltose within successive regions of the digestive tract (46). Similarly, while the stimulatory cue is unknown, after sensing changes in the environment within the host’s gastrointestinal tract, V. cholerae anticipates exiting the GI tract and induces genes that will aid in adjusting to changes in osmolarity and carbon source availability upon release into an aquatic environment (60). Yeasts also exhibit responses that could be categorized as adaptive prediction. Saccharomyces cerevisiae uses heat, acidity, and ethanol as early-fermentation cues to anticipate late- fermentation oxidative stress (46), and the pathogen Candida albicans mounts an anticipatory oxidative stress response upon exposure to glucose at concentrations present in the bloodstream, thereby enhancing resistance to stresses encountered within the host (57). In each case, the 65 response reflects the natural temporal order of changes in a specific environment and bestows enhanced fitness to the organism within that environment (45). Given the overlapping definitions and varied terminology used to describe these phenomena in the literature, we hereafter use the term “anticipatory response” for simplicity. The extent to which anticipatory responses are widespread beyond these examples is not known.
Daily fluctuations in light are a reliable characteristic of the Earth’s surface and provide both temporal and spatial information about the local environment. The diel light cycle is intrinsically tied to changes in other environmental factors, such as temperature and relative humidity, in terrestrial environments. These features make light a potentially useful signal for anticipatory responses. Many organisms, including microbes, are capable of sensing light signals using photosensory proteins, and these proteins regulate diverse phenotypes. In some photosynthetic organisms, light-signaling appears to be anticipatory in nature. For example, in
Arabidopsis thaliana plants, light primes for enhanced thermal detoxification of reactive oxygen species (23), and in desert crust cyanobacteria, light stimulates an anticipatory response against dehydration stress (50; 51). These examples reflect the potential coupling of light with heat and dehydration stress, respectively, and support the predictive nature of light as a signal for subsequent environmental stresses.
Terrestrial plant leaf surfaces are highly dynamic environments that undergo large changes in light exposure, temperature, and nutrient and water availability over the course of each day. Pseudomonas syringae pv. syringae B728a (Pss B728a) is a plant pathogen that is well adapted to living on leaf surfaces, even in the absence of disease (26; 27). Pss B728a contains three photosensory proteins: one blue light-sensing protein, designated LOV because of the presence of a light/oxygen/voltage (LOV) domain, and two red/far-red-light-sensing 66 bacteriophytochromes, designated BphP1 and BphP2 (5; 67). LOV and BphP1 are central components of a branched photosensory pathway in Pss B728a that regulates colonization, lesion size, and swarming motility (43; 73). As yet, no phenotypes have been attributable to BphP2. We previously discovered that light stimulates global transcriptional reprogramming of Pss B728a via BphP1 (Chapter 2). Notably, this reprogramming included the upregulation of a disproportionate number of genes involved in osmotic stress tolerance. Although light does not cause osmotic stress directly, osmotic stress mounts rapidly (< 5 min) in the phyllosphere following leaf exposure to drying conditions (1), and light and surface drying commonly occur at dawn. Accordingly, we predicted that Pss B728a cells on leaves may exploit light signals to anticipate a daily onset of drying, and an associated increase in osmolarity due to surface solutes, and thereby use these temporally coupled signals to enhance theirs fitness in the phyllosphere.
In agreement with this prediction, we demonstrate here that pre-exposure to red or far-red light enhanced the osmotolerance of Pss B728a. This anticipatory response required the red/far- red-light-sensing phytochrome BphP1, whereas other known components of the integrated
LOV/BphP1 photosensory pathway were not required. Thus, light-enhanced osmotolerance is regulated via a novel pathway downstream of BphP1. Light-enhanced osmotolerance also required the alternative sigma factor AlgU and proteins responsible for the import of osmoprotective compounds, revealing that light signaling is integrated with the direct osmotic stress response in Pss B728a. Finally, we discovered that light-enhanced osmotolerance is conserved among additional P. syringae strains and Pseudomonas species that inhabit the phyllosphere and rhizosphere. Taken together, these data reveal that plant-associated, light- sensing pseudomonads use light cues to prepare for subsequent osmotic stress and substantiate that light signals are used for anticipatory regulation in non-photosynthetic bacteria. 67
Methods
Bacterial strains and growth conditions
Strains and plasmids used in this study are listed in Table 1. Pseudomonas strains were routinely plated from glycerol freezer stocks onto King’s B (KB) agar (31) containing antibiotics at the following concentrations, as appropriate: rifampicin (50 µg/mL) and kanamycin (50
µg/mL). All osmotic stress studies were conducted in ½-21C broth (8) amended with 8.5 mM succinate (½-21CS). Liquid cultures were grown at 28°C with shaking at 200 RPM except for during light exposure, which was conducted statically at 18–20°C.
Table 4. Strains and plasmids used in this study
Strain or Plasmid Description Reference Strains B728a Wild-type P. syringae pv. (37) syringae; RifR ∆bphOP1 B728a ∆Psyr_3505-3504; RifR (73) ∆lov B728a ∆Psyr_2700; RifR (73) ∆bphP2R B728a ∆Psyr_2384-2385; RifR (73) ∆bsi B728a ∆Psyr_2699; RifR (43) ∆ahlR B728a ∆Psyr_1622::Km; RifR, (54) KmR ∆smp B728a ∆Psyr_2449::Km; RifR, (43) KmR ∆kaiC B728a ∆Psyr_2451; RifR L. Wu and G.A. Beattie, unpublished data ∆algU B728a ∆Psyr_3958; RifR (75) 1448A Wild-type P. syringae pv. (39) phaseolicola; RifR 302699 Pseudomonas cichorii (77) B301D-R Wild-type P. syringae pv. (19) syringae; RifR CHA0 Wild-type Pseudomonas (65) protogens HS191 Wild-type P. syringae pv. (18; 19) syringae KT2440 Wild-type Pseudomonas putida (2)
68
Table 4 Continued Strain or Plasmid Description Reference Strains BT B728a ∆Psyr_4249-4252, (11) ∆Psyr_4709-4711, ∆Psyr_4827; RifR ∆ggnA B728a ∆Psyr_3747; RifR (10) ∆ggnAB∆tre1∆tre2 B728a ∆Psyr_3747-3748, X. Yu and G.A. Beattie, ∆Psyr_2489-2491,_ unpublished data ∆Psyr_2992-3001::Km; RifR, KmR ∆betT B728a ∆Psyr_4827; RifR S. Li and G.A. Beattie, unpublished data B728a pN-bphOP1 B728a with plasmid pN (11) (73) carrying a constitutively expressed bphOP1 locus; RifR, KmR
Light exposure setup
LED light bars (730 nm [far-red light], 450 nm [blue light], and 660 nm [red light]) equipped with dimming switches were purchased from BML Horticulture (now Fluence
Bioengineering, Austin, TX). Intensities for bacterial light exposure were manually controlled with the dimming switches and by adjusting the distance of the bacterial cells from the light bar.
Wavelengths and exposure intensities were measured using a spectrometer (BLACK-Comet
CXR-100, StellarNet Inc., Tampa, FL). This setup enabled blue- and red-light intensities of 0–30
µmol/m2/sec and far-red light intensities of 0–10 µmol/m2/sec. For all analyses, light-exposed cultures were compared to paired dark controls that were wrapped in a double layer of aluminum foil and incubated under the same light bars.
Assessment of light impacts on bacterial osmotolerance based on optical density measurements
For each strain, replicate cultures inoculated from single colonies were grown overnight in ½-21CS and then sub-cultured into fresh ½-21CS for 4 h at 28°C to obtain exponentially growing cells. Cultures were then normalized to an optical density at 600 nm (OD600) of ~0.2 69 using a plate reader (SpectraMax iD3, Molecular Devices, LLC, San Jose, CA). For each replicate culture, 1 mL was placed into an individual well of two 6-well plates, and the plates were then exposed to light or maintained in the dark for times ranging from 15 min to 4 h, as specified in the figure legends. Immediately after light exposure, cells were exposed to high osmolarity by transferring 50 µl of cells to 150 µl of ½-21CS amended with NaCl (1.0 M final conc) and choline chloride (1 mM final conc) in a 96-well plate. For the multi-strain osmotolerance test, cells were transferred to ½-21CS amended with NaCl at 0.75 M or 1.0 M
(final concs) in the presence of glycine betaine and choline chloride (1mM final conc. each). Cell growth was monitored every 30 min for 20 h by measuring the OD600 in a SpectraMax iD3 plate reader; cultures were incubated at 28°C with continuous shaking over the 20 h. Following normalization of values to the OD600 at the initial time point (T0), the normalized values were plotted versus time and the area under the curve (AUC) values were calculated using the Area
Below Curves macro function in SigmaPlot 14.0 (Systat Software, Inc).
Assessment of light impacts on bacterial osmotolerance based on cell viability
Cell viability, assessed based on viable plate counts on KB agar, was monitored for cells subjected to the following four treatment schemes: light exposure with and without subsequent osmotic stress and cells kept in the dark with and without subsequent osmotic stress. Cultures were prepared as for the optical density experiments above, where NaCl (1.0 M) and choline chloride (1 mM) were present in all of the treatments. Care was taken to treat the cultures between the two types of experiments as similarly as possible. Cell densities were measured at
T0 using both a SpectraMax iD3 plate reader and by viable plate count on KB agar with
Rifampin. The 96-well plate was then incubated in the plate reader at 28°C with shaking for 2 h, at which time the cell densities were again determined by viable plate count. 70
Environmental monitoring preceding and following dawn
Environmental data were collected on Sept. 15, 2020, from the rooftop of Agronomy Hall on the Iowa State University campus in Ames, Iowa, USA (42.02838° N, 93.64248° W).
Measurements were taken over 6.5 h, starting approximately 90 min prior to sunrise.
Atmospheric humidity and temperature were measured every 10 min using a Watchdog A-series datalogger (Spectrum Technologies, Inc., Aurora, IL). Total light intensity was recorded approximately every 5-min using a BLACK-Comet CXR-100 spectrometer equipped with a
CR2-OR sensor (StellarNet, Inc.). Due to equipment constrains, the CR2 sensor was oriented perpendicular to the ground for the entirety of the experiment. As the sensor orientation did not track with the movement of the sun, the measured light intensities are likely underestimates. In addition, to stay within the dynamic range of the spectrometer, a CR-AP aperture (CR-AP,
StellarNet, Inc.) was added to the sensor once the minimum photon integration time (30 ms) yielded an intensity reading > 85% of the detector capacity; this occurred at approximately 8:40 am CST and addition of the aperture further exacerbated the underestimation of intensity readings. Ranges of intensity for blue, far-red, and red light were extracted from the recorded data by limiting the integration range within the software to 400-500 nm for blue light, 600-700 nm for far-red light, and 680-780 nm for red light. These ranges were determined by recording the spectra from our LED light bars and defining a 100-nm range of wavelengths centered on the peak of each LED light bar.
Dew formation and evaporation were measured using filter discs (Whatman no. 1, 90-mm diameter) as hydrophilic surfaces and weigh boats (Fisher small square polystyrene weigh boats) as hydrophobic surfaces using a destructive sampling approach. Specifically, 150 μL of sterile nano-pure water was placed on the center of each pre-weighed filter disc in 100 x 100-mm square petri dishes, with four filter discs per dish, immediately prior to the start of the 71 experiment. Similarly, 400 μL of water was placed onto the center of each weigh boat in similar square petri dishes, with four weigh boats per petri dish, immediately prior to the start of the experiment; these were minimally disturbed to maximize uniformity in shape and surface area of the droplets. All petri dishes were kept covered until T0, at which point all lids were removed.
The filter discs and weigh boats (four replicates per type per time point) were weighed using a
NewClassic MF scale (Model: MS8001S, Mettler Toledo) at intervals ranging from 5 min to 20 min, with the shorter intervals used during periods when evaporation was particularly rapid. For each time point, the weight of the water of a given sample was calculated by subtracting the pre- weight of the filter disc or weigh boat from the measured sample weight. The smoke from the
2020 California and Colorado forest fires may have been present in the skies during this test, but we do not believe that this substantially affected our readings or altered our conclusions. This test was also performed on Oct. 17, 2019, with similar results in the rate of evaporation, but the light readings were accurate only for the hours prior to needing an aperture on the spectrometer
(i.e., only for the first 2 to 3 hours).
Statistical Analysis
One-way and two-way analyses of variance (ANOVAs) were performed using the software program JMP Pro 15 (SAS Institute, Inc) with treatment conditions and either experimental replicate (when replicated experiments were combined for analysis) or strain as factors. If the ANOVA reported a p ≤ 0.05, the p-value is reported, and multiple comparisons were performed using Tukey’s HSD with a p ≤ 0.05 to evaluate differences among strains and treatments. For the multi-strain osmotolerance test, Student t-tests (paired, two-sided) were performed using Microsoft Excel to evaluate the difference in AUC values between light- and dark-exposed samples for a given strain. 72
Results
Pre-exposure to far-red or red light enhances osmotolerance of Pss B728a.
We previously observed that a 15-min exposure to far-red, red, or blue light induced expression of genes involved in osmotolerance in Pss B728a (Chapter 2). To test whether light exposure influenced osmotolerance, we exposed Pss B728a cultures to far-red, red, or blue light for 30 min or 1, 2, or 4 h and then exposed the cells to media containing 1 M NaCl and choline as an osmoprotectant. We examined multiple durations of exposure to light to evaluate if the cellular response depended on light signal duration. After plotting growth based on OD600 versus time for cultures pre-exposed to far-red light (Fig. 1A) or red light (Fig. 1B), the area under the curve (AUC) was used as a measurement of the total growth of a culture under a given condition.
In agreement with our hypothesis, pre-exposure to light led to enhanced osmotolerance.
Specifically, all tested pre-exposure durations to far-red and red light enhanced the osmotolerance of Pss B728a compared to a 4-h exposure to the dark. A 1-h pre-exposure to far- red light was sufficient to yield maximal tolerance (Fig. 1C), with red light inducing a lower level of osmotolerance than far-red light (Fig. 1D). Although a 15-min pre-exposure to blue light was sufficient to induce the expression of osmotolerance-related genes in Pss B728a
(Chapter 2), we did not detect a consistent anticipatory blue light-mediated enhancement in osmotolerance. We did find that a 4-h pre-exposure to blue light was detrimental to culture growth upon subsequent osmotic shock (data not shown), which is in line with blue light leading to cell stress (13; 21; 22). Overall, these results demonstrate that pre-exposure to far-red or red light for as little as 30 min induces enhanced osmotolerance in Pss B728a; we have seen anticipatory light-enhanced osmotolerance after a shorter induction period of 15-min pre- exposure (data not shown) which is in line with changes in gene expression following a 15 min 73 light exposure (Chapter 2). As far-red light provoked the highest degree of protection, we used far-red light for all subsequent assays examining light-enhanced osmotolerance.
Figure 1. Pre-exposure to (A, C) far-red light or (B, D) red light confers enhanced osmotolerance to Pss B728a. After exposure of cells to dark, far-red light (10 μmol/m2/sec), or red light (30 μmol/m2/sec) for the indicated duration, cells were exposed to 1 M NaCl in the presence of 1 mM choline and growth was monitored based on optical density at 600 (OD600). The growth curves for individual cultures of two representative treatments are shown for the far-red (A) and red (B) light experiments. Growth curves were normalized by dividing every OD600 measurement by the OD600 reading at T0 for each individual replicate. Area under the curve (AUC) values were derived for each culture, and these values reflecting total growth are shown as the mean AUC and standard error of the mean (SE) from 12 replicates (n = 6 in each experimental replicate) for each exposure duration (C,D). Statistical significance was assessed using a two-way ANOVA with experimental replicate and exposure duration as factors (A: p < 0.0001, B: p = 0.004). Letters indicate significant differences among treatments determined using Tukey’s HSD at p < 0.05.
74
The magnitude of anticipatory light-enhanced osmotolerance is not strongly dependent on light-intensity
We previously observed that higher intensities of light stimulated higher expression levels of the light-responsive gene algD (Chapter 2). Thus, we postulated that anticipatory light- enhanced osmotolerance may similarly depend on light intensity, or alternatively, it may be a binary response in which pre-exposure to any light intensity stimulates the same level of enhanced osmotolerance. To distinguish between these possibilities, we evaluated the impact of a
30-min pre-exposure to 2, 5, or 10 µmol/m2/sec far-red light on tolerance to 1 M NaCl with choline. All tested light intensities conferred enhanced osmotolerance compared to no pre- exposure (maintenance in the dark), with no statistical differences between the growth after pre- exposure to the three light intensity levels (Fig. 2), although the magnitude of the enhancement trended toward better protection at higher light intensities. From these results, we cannot differentiate between the two proposed models (Fig. 2). 75
Figure 2. Anticipatory far-red light-enhanced osmotolerance is not clearly intensity dependent. Cultures of Pss B728a were exposed to the dark or to the indicated intensities of far-red light for 30 min prior to challenge with 1 M NaCl in the presence of 1 mM choline. Bars represent the mean AUC and SE from 18 replicates (n = 6 for each of three replicate experiments) for AUC values generated as described in Fig. 1. A two-way ANOVA with experimental replicate and pre-exposure duration as factors was used for analysis (p < 0.0001). Letters indicate significant differences among treatments determined using Tukey’s HSD at p < 0.05.
The photosensory protein BphP1 is required for anticipatory light-enhanced osmotolerance in Pss B728a
Loss of the Pss B728a bphOP1 locus, which encodes the bacteriophytochrome BphP1 and the heme oxygenase required to make the chromophore, eliminated the light-mediated regulation of almost all osmotic stress tolerance genes in an RNAseq study (Chapter 2). Here, using the ∆bphOP1 mutant, we found that BphP1 was absolutely required for anticipatory light- enhanced osmotolerance in Pss B728a (Fig. 3A). That is, regardless of pre-exposure to light, the
∆bphOP1 mutant exhibited osmotolerance comparable to that of wild type (WT) maintained in the dark. Anticipatory light-enhanced osmotolerance was fully restored by complementation of the mutant with bphOP1 expressed from a plasmid. These results establish that BphP1 is critical to the ability of Pss B728a to use light signals to enhance its fitness during a subsequent exposure to osmotic stress. 76
Next, we were interested in placing anticipatory light-enhanced osmotolerance into the mapped photosensory pathway of Pss B728a (43; 44; 73). We tested if any known or hypothesized proteins in the mapped photosensory pathways are involved in anticipatory light- enhanced osmotolerance by testing mutants deficient in selected loci in the same manner as described above (Fig. 3B). The algU gene was required for Pss B728a growth under hyperosmotic conditions, as evidenced by the lack of growth of the ∆algU mutant regardless of light pre-exposure (Fig. 3C, 3E), as expected since it encodes a sigma factor critical for the osmotic stress response (32; 62; 71; 75). Of the known pathway components (43; 44; 73), namely smp, lov, ahlR and bsi, none were required for anticipatory light-enhanced osmotolerance based on that each mutant grew better under hyperosmotic conditions after pre-exposure to light than in the dark (Fig. 3B). Two additional loci of interest were bphP2R and kaiC, which encoded a second bacteriophytochrome (BphP2) and its predicted response regulator (BphR), and a circadian clock regulatory protein (KaiC), respectively. BphP2R and KaiC similarly were not required for anticipatory light-responsive osmotolerance (Fig. 3B). However, differences in growth between the WT and several mutants suggest that these components play novel roles in osmotolerance in Pss B728a. For example, the ∆smp mutant grew better than the WT after both pre-exposure to the light and maintenance in the dark (Fig. 3B, 3D, 3E), indicating that Smp negatively impacts Pss B728a osmotolerance in a light-independent manner. In contrast, the
∆bphP2R and ∆ahlR mutants grew slightly less than the WT when maintained in the dark (Fig.
3B, 3E) but equivalently to the WT when pre-exposed to light (Fig. 3B, 3C), resulting in larger differences between the growth in the presence versus absence of light for these mutants than for 77
Figure 3. BphP1 and AlgU are involved in anticipatory light-enhanced osmotolerance. (A) Growth of B728a, a bphOP1 deletion mutant, and a complement of this mutant, and (B) growth of B728a and selected isogenic mutants, upon challenge with 1 M NaCl in the presence of choline with or without a 30-min pre-exposure to 10 µmol/m2/sec far-red light; growth is shown as area under the curve (AUC) values, and the data were analyzed as described in Fig. 1. (C) For individual cultures in (B), the difference in AUC values between growth with and without light pre-exposure was determined, with these difference values analyzed as in Fig. 1. (D, E) Average growth curves from one replicate experiment in (B) in which cultures were (D) pre-exposed to light or (E) maintained in the dark (n = 5 for the WT and n = 6 for the mutants). Bars represent the mean and standard error (SE) from (A) 10 replicates (n = 5 for two replicate experiments), (B, C) 10 replicates for Pss B728a WT (n = 5 for two replicate experiments) and 12 replicates for all mutant strains (n = 6 for two replicate experiments). (A, B, C) Letters indicate significance as calculated via a multiple-comparisons analysis with a Tukey’s HSD p < 0.05.
78 the WT (Fig. 3D). These findings suggest that BphP2 and/or BphR and AhlR are beneficial to osmotolerance in dark-maintained cells. While the mechanism behind this loss of growth in dark maintained cultures is unknown, it may be that BphP2 senses the absence of light, as suggested for another photosensory protein (59), and both BphP2 and/or BphR and AhlR influence traits that affect growth. The lack of impact of BphP2/BphR on growth in rich culture media without osmotic stress (73) and the impact of AhlR on epiphytic growth following drying conditions (54) indicate that the impacted traits are relevant specifically under hyperosmotic conditions. Notably, this represents the first phenotype to be associated with the BphP2 protein in Pss B728a. Taken together, these results demonstrate that i) anticipatory light-enhanced osmotolerance is a novel phenotype controlled by the BphP1, ii) previously identified or predicted genes in the BphP1 photosensory pathways are not directly regulating anticipatory light-enhanced osmotolerance, and iii) some proteins in the photosensory pathway, namely Smp, BphP2 and AhlR, play a role in light-independent regulation of growth under conditions of osmotic stress.
Anticipatory light-enhanced osmotolerance requires the uptake and/or synthesis of osmoprotective compounds
Our prior RNAseq results revealed that osmoprotectant transporter genes and compatible solute synthesis genes were among those most strongly upregulated by light in a BphP1- dependent manner (Table 2). To evaluate the role of these functions in anticipatory light- enhanced osmotolerance, we evaluated anticipatory light-enhanced osmotolerance in four mutant strains: BT, which lacks all choline/glycine betaine osmoprotectant transporters, ∆ggnA, which lacks the ability to synthesize the compatible solute N-acetylglutaminylglutamine amide
(NAGGN), ∆tre1∆tre2, which lacks the ability to synthesize trehalose, and ∆ggnA∆tre1∆tre2, which is defective in both NAGGN and trehalose synthesis. We found that strain BT was completely deficient in anticipatory light-enhanced osmotolerance (Fig. 4); therefore, 79 osmoprotectant transport was required for anticipatory light-enhanced osmotolerance in the presence of an exogenous osmoprotectant. This finding is consistent with the preferential use of external osmoprotectants over de novo synthesis due to the lower energy needs for transport than biosynthesis (34). Strains ∆ggnA and ∆ggnA∆tre1∆tre2 were reduced in anticipatory light- enhanced osmotolerance, with intermediate AUC values between those of the WT and the BT mutant (Fig. 4). Thus, NAGGN synthesis contributed to anticipatory light-enhanced osmotolerance but was not absolutely required, as the uptake of the external osmoprotectants likely enabled enough compatible solute accumulation for growth but not enough for wild-type levels of growth. Because the AUC values for the ∆ggnA and ∆ggnA∆tre1∆tre2 mutants were comparable, we conclude that the contribution of trehalose synthesis under these conditions was minimal. Collectively, these data support a model in which light promotes the formation of osmoprotectant transporters prior to osmotic stress exposure. In particular, the transporter deficiency of the BT mutant, which lacks the choline/glycine betaine transporters OpuC (8),
BetT (9) and Cbc (11), eliminates the ability to uptake compounds that can either be used as compatible solutes, such as with glycine betaine (not tested here), or converted to compatible solutes, as when exogenous choline is brought in and converted to glycine betaine. Similarly, mutants lacking ggnA are unable to accumulate NAGGN by de novo synthesis (34). Thus, light exposure likely promotes biochemical priming of membrane transporters, which increases the speed of osmoprotectant import and therefore cellular maintenance of homeostasis upon exposure to an osmotic upshift in the presence of an osmoprotectant. In this case, the transporters would have enabled the rapid uptake of choline, which was provided at the time of the osmotic upshift, following a limited duration exposure to light. Taken together, our data places 80 anticipatory osmotolerance into a new branch within the BphP1-regulated integrated signaling network (Fig. 5).
Table 2. Osmoprotectant-related genes and their fold changes upon far-red light exposure in the wild type (WT) Pss B728a and in a bacteriophytochrome-deficient mutant
Gene Gene WT Far-red ΔbphOP1 Far-red Mechanism Number Name vs WT Dark vs WT Far-red Psyr_4249 opuCA Choline/betaine/carnitine transport 4.1 -4.2 Psyr_4250 opuCB Choline/betaine/carnitine transport 3.6 -4.2 Psyr_4251 opuCC Choline/betaine/carnitine transport 4.0 -4.4 Psyr_4252 opuCD Choline/betaine/carnitine transport 4.0 -4.4 Psyr_4709 cbcX Choline/betaine/carnitine transport 2.7 -2.8 Psyr_4710 cbcW Choline/betaine/carnitine transport 2.7 -3.0 Psyr_4711 cbcV Choline/betaine/carnitine transport 2.7 -2.8 Psyr_4827 betT Choline transport 2.5 -2.8 Psyr_3747 ggnA NAGGN synthesis genes 6.9 -6.6 Psyr_2490 treS Trehalose synthesis 4.6 -4.4 Psyr_2995 treY Trehalose synthesis 4.8 -4.3
Figure 4. Osmoprotectant transport is required for light-enhanced osmotolerance. Growth of Pss B728a and mutants deficient in choline transport (BT), NAGGN synthesis (ΔggnA), and NAGGN and trehalose synthesis (ΔggnAΔtre1Δtre2) upon challenge with 1M NaCl in the presence of choline with or without a 30-min pre-exposure to 10 µmol/m2/sec far-red light. Data are from a single experiment but are representative of two independent experiments. Analysis was conducted using a one-way ANOVA with strain as the treatment factor (p < 0.0001). Significance was assessed using Tukey’s HSD (p < 0.05). Bars represent the mean and SE from 6 replicates.
81
Figure 5. BphP1-regulated integrated signaling network in Pss B728a. This network has been expanded and modified from a pathway developed in (43). Grey arrows indicate genes that exhibit transcriptional regulation; colored boxes indicate proteins or signal compounds that are regulated.
Pre-exposure to light does not improve cell viability immediately following osmotic upshift
Whereas compatible solute accumulation confers enhanced adaptation to continued hyperosmolarity, a rapid influx of potassium ions is generally used to prevent water efflux in the period immediately following an osmotic upshift. This influx is mediated, in part, through transcriptional regulation of the Kdp potassium transport system; however, this system was not regulated by light, BphP1 or AlgU (Chapter 2). We hypothesized that the light-mediated priming to accumulate compatible solutes could improve tolerance to the rapid water efflux immediately following an osmotic upshift, and that this would be evident in improved cell viability during this upshift. To test this hypothesis, we measured the cell viability for Pss B728a cells that were or were not pre-exposed to far-red light for 30 min immediately following an osmotic upshift in the presence of choline, 2 h after this upshift, or at 0 and 2 h of exposure to non-stressed conditions.
Cell viability was not influenced by pre-exposure to light based on that a 2-h osmotic upshift resulted in a >10-fold decrease in colony forming units (CFUs) on KB agar regardless of light pre-exposure (Fig. 6). Unfortunately, because cultures subjected to osmotic shock contained a 82 high proportion of dead cells (Fig. 6), which distorts OD-based growth measurements, we were unable to determine if light pre-exposure influenced the growth rate of Pss B728a during osmotic stress. However, because cultures pre-exposed to light consistently achieved higher densities than dark-maintained cultures within the 20-h monitoring period (Fig. 1A), but cell viability following the initial transfer to a high osmolarity medium was not influenced by light (Fig. 6), the anticipatory light-enhanced osmotolerance reflected either a decreased lag time, a faster growth rate, or a combination of the two.
Figure 6. Light-enhanced osmotolerance is not due to increased viability immediately following osmotic upshift. Cells of Pss B728a were kept in basal medium (no osmotic stress) or were subjected to osmotic upshift following a 30-min pre-exposure to far-red light or maintenance in the dark before a viability assessment based on growth on KB agar with rifampin. Analysis was conducted using a one-way ANOVA (p < 0.0001). Significance was assessed using Tukey’s HSD (p < 0.05). Bars represent the mean and SE from 6 replicates.
The onset of light at dawn precedes dew evaporation
To better understand the timing between the onset of light and the loss of water from a leaf surface in the morning hours, we collected environmental data (temperature, humidity, and dew point) from dawn to 1:00 pm (Fig. 7A) while measuring light intensity, dew accumulation, and evaporation using plastic weigh boats to simulate hydrophobic leaf surfaces and filter paper 83 discs as highly hydrophilic surfaces (Fig. 7B). Dew accumulated in addition to the water that was placed on the weigh boats and filter paper discs, illustrating the environmental fluctuations in the early morning. Dew accumulation is a non-meteorological event that occurs daily across a range of environments, even during drought (17). Light from 300 nm to 800 nm was measurable for more than 1.5 h prior to any measurable loss of water (Fig. 7B). We observed the same trend in measurements taken in the same location on Oct. 17, 2019 (data not shown). This finding demonstrates that, at least under some conditions, the phyllosphere and its bacterial residents are exposed to light prior to the onset of evaporative water loss. The appearance of light prior to drying is consistent with our demonstration of anticipatory light-mediated osmotolerance, as osmotic stress would accompany evaporative drying from surfaces where leaf-derived nutrients and other solutes are present.
We showed that far-red light intensities as low as 2 µmol/m²/sec were sufficient to activate anticipatory light-enhanced osmotolerance in Pss B728a (Fig. 2). Although the actual light intensities that reach the Earth’s surface depend strongly on geographic location and season, here we detected light intensities capable of inducing this biological activity long before we observed measurable water loss from exposed surfaces. We predict that this temporal sequence of events is common to many terrestrial surfaces. Our recording of measurable light intensities prior to evaporative water loss in the early morning, taken together with our anticipatory light- enhanced osmotolerance data, support that Pss B728a cells sense light early in the morning and use that light, particularly far-red light, as a signal to prime against oncoming osmotic stress. 84
Figure 7. Light exposure preceded evaporative water loss from surfaces exposed to sunlight. (A) The relative humidity, temperature, and dew point, and (B) the intensities of natural light over the wavelength ranges for blue light (400-500 nm), red light (600-700 nm), and far-red light (680-780 nm) and the accumulation and evaporation of surface water from hydrophobic and hydrophilic surfaces, from 6 am to 1 pm on September 15, 2020, in Ames, Iowa. The shaded box represents the period when light was present prior to evaporation onset. The asterisk indicates the addition of an aperture to the light sensor to ensure accurate readings at high light intensity levels. The weight of the water present on weigh boats (hydrophobic surfaces) was normalized to the maximum water weight present on filter discs (hydrophilic surfaces) to aid in visualization in the figure. The values shown for relative humidity, temperature, and dew point are the mean and SE for 3 replicates. The values shown for surface water are the mean and SE for 4 replicates.
Anticipatory light-enhanced osmotolerance is conserved in several Pseudomonas spp.
Because of the temporal relationship of light onset to drying on leaves (Fig. 7), Pss
B728a may have developed a light-primed preadaptation response to predictable oncoming osmotic stress, an adaptation that could greatly increase its epiphytic fitness. We evaluated if other plant-associated bacteria exhibit a similar fitness advantage from light sensing. We selected five additional Pseudomonas spp. strains (Table 3), including three that are generally associated 85 with leaves (two P. syringae strains and a P. cichorii strain), and a couple that are generally associated with roots or soil (a P. protegens strain and a P. putida strain). The genomes of these bacterial strains all encode at least one bacteriophytochrome protein, but collectively these proteins vary in their similarity to Pss B728a BphP1 (Table 3). Two commonly studied P. syringae strains, Pst DC3000 and P. savastanoi pv. phaseolicola 1448A, did not grow in the low osmoticum medium used for this study, ½-21CS, and thus were not included. The selected strains were tested for osmotolerance following pre-exposure to light as was done for Pss B728a in Figs. 3-5 above, but with three modifications. First, cultures were pre-exposed to far-red light for 2 h rather than 30 min to give ample time for light-induced cellular responses to occur.
Second, following pre-exposure, the strains were challenged with two NaCl concentrations (0.75
M or 1.0 M) to accommodate variation in their natural levels of osmotolerance. Third, the growth medium was amended with 100 mM glycine betaine in addition to 100 mM choline chloride, as glycine betaine is the preferred osmoprotectant for many bacterial species (6; 20).
Table 3. Characteristics of pseudomonads tested for anticipatory light-enhanced osmotolerance
Strain Species Habitat % amino acid identity of phytochrome to Pss B728a BphP1 B728a P. syringae pv. syringae Phyllosphere 100 B301D-R P. syringae pv. syringae Phyllosphere 99.5 HS191 P. syringae pv. syringae Phyllosphere 97.2 302699 P. cichorii Phyllosphere 80.4 CHA0 P. protegens Roots and soil 72.8 KT2440 P. putida Roots and soil 34
All five Pseudomonas spp. strains tested exhibited light-enhanced osmotolerance in one or both salt concentrations. Strains P. protegens CHA0 and P. cichorii 302699 exhibited significant light-enhanced osmotolerance at 0.75 M NaCl (Fig. 8A); the lack of an impact of 86 light at 1 M NaCl may reflect that this NaCl concentration is above their tolerance threshold.
Strains Pss HS191 and Pss B301D-R were similar to Pss B728a in exhibiting light-enhanced osmotolerance at 1 M NaCl, further confirming conservation of the light-mediated phenotype.
The lack of light-enhanced osmotolerance by these three Pss strains at 0.75 M NaCl (Fig. 8B) may be because this concentration was not particularly stressful, as reflected in their strong growth irrespective of light. Unexpectedly, light pre-exposure reduced the growth of Pss HS191 and Pss B301D-R at 0.75 M NaCl when compared to the lack of light pre-exposure, with Pss
B728a showing a similar trend (Fig. 8A). These results suggest that, in the absence of a sufficient stress, light exposure can incur a fitness cost, in this case potentially by promoting the allocation of resources away from culture growth and to activities (transport and biosynthesis) that are not needed and thus waste resources. The last strain, P. putida KT2440, has the bacteriophytochrome with the least similarity to Pss B728a BphP1, but still exhibited light-enhanced osmotolerance, and did so at both NaCl concentrations (Fig. 8A, B). Given that P. putida KT2440 and P. protegens CHA0 are considered to be root- and soil-dwelling bacteria, these results suggest that light may serve as a useful signal for controlling osmotolerance in terrestrial environments beyond the phyllosphere. Overall, these results demonstrate that anticipatory light-enhanced osmotolerance is a conserved trait among all of the Pseudomonas species that were examined. 87
Figure 8. Light-enhanced osmotolerance is conserved among Pseudomonas species. Growth of selected Pseudomonas strains that were or were not pre-exposed to far-red light for 2 h prior to their transfer to media with (A) 0.75 M or (B) 1.0 M NaCl and 100 mM glycine betaine and 100 mM choline. Student’s t-tests (two-tailed) were done to compare differences in the AUC values derived from cultures grown with and without pre-exposure to light. * indicates p-value <0.09, ** indicates p-value <0.05. For all samples n = 3.
Discussion
Here, we demonstrated that Pss B728a uses light as a signal to induce tolerance to subsequent exposure to osmotic stress, a phenomenon we designate as anticipatory light- enhanced osmotolerance. We demonstrated that this phenomenon occurs in all three of the P. syringae strains tested, and in strains of all four Pseudomonas species tested, indicating that this is a conserved trait. Bacteriophytochromes are present in over half of the 92 represented
Pseudomonas spp. and 75% of the strains among the 747 strains with complete genome sequences in the Pseudomonas database (72), supporting the potential for widespread conservation among pseudomonads. This conservation could extend further, as bacteriophytochromes are present in diverse bacterial taxa, both Gm- and Gm+ (38), but we have not yet explored this possibility or whether the conservation exists in non-plant-associated pseudomonads. 88
Pre-exposure to red light or far-red light for as little as 15 min enhanced Pss B728a growth under osmotic stress conditions in comparison to cells that were not pre-exposed to light.
This rapid response is shorter than the delay between the first appearance of light and the onset of evaporative drying on exposed surfaces that we observed in a temperate terrestrial environments; we showed this temporal delay to be over 1.5 hours on a selected fall day in
Iowa. We predict that, because of the low intensities required to activate the response, most environments exhibiting evaporative surface drying will similarly experience light exposure prior to the onset of drying. Notably, far-red light conferred a greater degree of protection against osmotic stress than did red light. This finding agrees with our prior results that far-red light is a particularly potent light signal for altering gene expression in Pss B728a (Chapter 2) and supports the model that far-red light is an effective signal to preemptively mount protective responses against ensuing osmotic stress.
We were somewhat surprised to find that P. putida strain KT2440, a pseudomonad that thrives in soil and root environments (47) and harbors a bacteriophytochrome with a low similarity to PssBphP1, also exhibited anticipatory light-enhanced osmotolerance. This phenomenon could thus be widespread not only among phyllosphere-residing microbes but also among those residing in the soil and rhizosphere. Interestingly, accumulating evidence indicates that ambient light may reach these sites. Light can penetrate as deep as 5–7 mm into the soil though the spaces separating soil aggregates (52; 69), exposing bacteria on the surfaces of these upper soil aggregates to light. Light can also penetrate deeper into the soil through soil fissures that occur during periods of drying and drought (74). Notably, under canopies, the light penetrating the soil is enriched in far-red wavelengths due to filtering of other wavelengths by the canopy above (64). Light can also be piped through plant stems into the roots via the plant 89 vasculature (42; 49; 66), with far-red wavelengths again being enriched due to their lack of absorption by photosynthetic pigments (66). To date, the question of whether piped light exits the roots and penetrates the surrounding soil has not been answered, but given that piped far-red light penetrates the root tissue surrounding the vasculature (66), low levels of far-red light likely escape from the roots into the surrounding soil enabling interception by root and soil bacteria.
Many root-associated bacteria have genes for bacteriophytochromes and may be subject to diel oscillations in water availability. For example, Agrobacterium (tumefaciens) fabrum and
Azospirillum brasilense are root colonists whose genomes each code for two bacteriophytochromes that are functional based on their regulation of conjugation and oxidative stress tolerance, respectively (3; 33). The same diel patterns in solar radiation and temperature that promote leaf surface drying can also affect soil drying, particularly in surface soils where the region of soil that is penetrated by light also experiences fluctuating moisture content (7; 36; 61).
These diel patterns are likely to be even more pronounced around roots, where sunlight can penetrate channels in the rhizosphere soil and potentially radiate out from the root following conductance through the plant. Diel fluctuations in the water content surrounding roots are also driven by the transpiration stream, which draws water to the roots because of evaporation in the phyllosphere and thus is light-dependent.
Prior studies with other microbes have observed elements of anticipatory light-enhanced osmotolerance. In the fungal species Aspergillus nidulans, the phytochrome AnFphA participates in the SakA-signaling pathway responsible for osmotic-stress sensing (76), but the effect of light on osmotolerance was not tested. In the cyanobacterial species Leptolyngbya ohadii that lives in desert soil crusts, light exposure promotes protection against desiccation, and phytochromes and cryptochromes have been postulated but not yet demonstrated to have a role; however, far-red 90 light exposure decreased rather than increased desiccation tolerance (50; 51). Lastly, in the β- proteobacterial strain Ramlibacter tataouinensis isolated from a desert sand, far-red and red-light exposure promotes the transition of cells from a non-motile to a motile form. This light regulation is mediated by two bacteriophytochromes, with the light regulation of the cell cycle predicted to promote rapid division and dissemination in the early morning when water from dew accumulation on desert soils is available. The subsequent onset of surface drying triggers the formation of non-motile, desiccation-tolerant cysts (12). Whereas the R. tataouinensis study is consistent with light as a signal to anticipate subsequent desiccation conditions, the organism was not evaluated for an anticipatory response; that is, light and water were studied as co- occurring signals in the study. Although these studies do not provide direct evidence of anticipatory responses that enhance tolerance to drying, collectively, the findings suggest that many terrestrial microbes are likely capable of exploiting the temporal coupling of light and surface water loss to enhance survival.
This study identified a new branch of the BphP1 light-sensing pathway in Pss B728a, a branch that integrates light signals with a direct stress-sensing pathway (4; 43; 44; 73). The requirement of BphP1 for light-enhanced osmotolerance was expected given that BphP1 is responsible for global transcriptional reprograming in response to light, including that of osmotolerance-related genes (Chapter 2). Light-enhanced osmotolerance also required AlgU, a broadly conserved sigma factor involved in stress responses in various pseudomonads (30; 32;
40; 41; 63; 71; 75). A mutant lacking algU did not tolerate osmotic stress regardless of light exposure. Notably, the algU gene is in the light-dependent, BphP1 regulon (Chapter 2), but
BphP1-mediated algU regulation must involve another regulatory protein since BphP1 lacks a
DNA-binding domain. Unfortunately, the identity of such regulatory proteins remains a mystery. 91
None of the known cellular components in BphP1-regulated pathways were required for anticipatory light-enhanced osmotolerance, indicating that this response is regulated via a novel pathway within the BphP1 regulatory network, a pathway that involves AlgU.
The integration of phytochrome-mediated light signaling into stress response pathways is emerging as a common mechanism for photoregulation and likely enhances cellular fitness. For example, the phytochrome AnFphA in A. nidulans signals via a stress-activated kinase (SakA) that mediates responses to both osmotic and oxidative stresses (16; 29; 70). In Pseudomonas aeruginosa, light-activated PaBphP1 phosphorylates the response regulator AlgB, which leads to repression of biofilm formation; this same regulator also integrates quorum sensing signals (48).
Biofilm formation can play an important role in the osmotic stress response (15; 53), and in Pss
B728a, the genes responsible for synthesis of the biofilm matrix exopolysaccharide alginate are upregulated in response to both osmotic up-shock (15) and light via BphP1 (Chapter 2).
Additionally, the acyl homoserine lactones (AHLs) that mediate quorum sensing in Pss B728a affect swarm motility in a light- and BphP1-dependent manner (43). The genes algB, kinB, and others involved in biofilm formation may be part of the PssBphP1 photosensory pathway and will be targets of future testing for involvement in light-enhanced osmotolerance. Taken together, the integration of light as an anticipatory signal to initiate direct stress response pathways could enhance fitness beyond what is provided by a direct sense-and-response strategy alone.
Light may also trigger anticipatory responses against other stresses in Pss B728a. For example, light upregulated genes involved in the oxidative stress response (Chapter 2). The reactive oxygen species (ROS) that exert oxidative stress are generated endogenously via electron transfer to oxygen (28). ROS are prevalent in the phyllosphere where plants utilize ROS 92 as both a direct defense against bacterial invasion as a signal within the plant for stimulating defense pathways (14). Plants have been shown to mount more effective defense responses in the light than in the dark (58), and ROS production has been found to be regulated on a diurnal cycle in Arabidopsis, with production peaking in the morning (35). Thus, oxidative stress is another stress that would be predicted to be temporally coupled to light onset in the phyllosphere and thus would be logical to regulate in an anticipatory manner in Pss B278a and other phyllosphere- residing microbes.
In conclusion, our discovery of light-enhanced osmotolerance advances our understanding of both photo-regulated physiology and anticipatory adaptive responses in bacteria. With the addition of light-enhanced osmotolerance to the repertoire of BphP1-regulated phenotypes, BphP1 is emerging as a major potential contributor to Pss B728a fitness in the phyllosphere. Although a recent study did not find a role for BphP1 in Pss B728a fitness on leaves (24), the study was conducted under conditions of high humidity in a growth chamber; together the high humidity and environmental control modulated by the growth chamber could have eliminated measurable contributions of anticipatory light-enhanced osmotolerance to fitness. The conservation of anticipatory light-enhanced osmotolerance in several plant- associated pseudomonads suggests a benefit of this anticipatory response on plant surfaces and in other environments in which light onset and osmotic stress are temporally coupled. Our results also raise fundamental questions regarding if bacteria have evolved to exploit other signals that are temporally coupled with light in terrestrial habitats, and what mechanisms these microbes might use to exploit light as an anticipatory cue to enhance fitness.
Acknowledgements
We are grateful to Chiliang Chen, Regina McGrane, Liang Wu, Xilan Yu, and Shanshan
Li for the construction of mutants that were used in this project, and to George Sundin and Larry 93
Halverson for providing strains. This project was supported by the Agriculture and Food
Research Initiative competitive grants program, Award number 2010-65108-20562 and 2015-
67013-23005 from the USDA National Institute of Food and Agriculture.
References
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CHAPTER 4. GENERAL CONCLUSIONS
Photosensory proteins are present in a range of species and are particularly common in plant-associated bacteria (10). This thesis was focused on improving our understanding of the signal transduction pathways involved in photosensing in B728a as a model organism. By conducting transcriptome analyses with the wild type, photosensory protein mutants, and complemented derivatives as they responded to various wavelengths of light, we aimed to identify the genomic, functional, and regulatory components of the P. syringae B728a response to light. Here, I provide some final thoughts on our progress in understanding the photosensory signal transduction pathways in this organism.
BphR as a potential response receiver for BphP1
Light and BphP1 have genome-wide effects on transcription in B728a, but surprisingly
LOV had very little effect. This suggests that LOV may primarily function post-transcriptionally in regulating the few phenotypes that it has been associated with in B728a (16; 17; 27). We also did not find that the loss of the second phytochrome and response regulator, BphP2 and BphR, affected the transcription of any genes under the conditions tested. The inability to detect BphP2- regulated genes may be because we did not use the optimal mutant to test this. We compared a
ΔbphOP1 mutant to a ΔbphOP1ΔbphP2R mutant with the goal of identifying those genes regulated by BphP2 independently of BphP1, but we did not find genes that differed in expression between the mutants. However, we found unexpectedly that bphR appears to be regulated separately from bphP2, rather than being in an operon as we had predicted. Moreover, bphR but not bphP2 was regulated by light in a BphP1-dependent manner. Based on the BphP1- dependent regulation of bphR, and the fact that BphP1 lacks a response regulator, BphR may be a functioning response regulator for BphP1. If BphR relies on phosphotransfer from BphP1, 99 there would not be differences in the regulons of the two mutants that were tested due to that
BphP1 was disabled in both. Two ways to test the theory that BphR functions as a response receiver for BphP1 include first, a phosphotransfer assay to determine if BphP1 can transfer a phosphate group to BphR, and second, qRT-PCR to compare the expression of genes known to be regulated by light and BphP1 in wild type B728a, ∆bphP1, and ∆bphR under light and dark conditions. The finding that the independent loss of BphP1 and BphR lead to similar changes in gene regulation patterns when compared to the wild type would confirm that the two proteins function in the same pathway.
While we were unable to identify changes in gene expression from the loss of the bphP2- bphR locus, we did identify a possible altered growth phenotype of theΔbphP2R mutant in a minimal medium (1/2-21C amended with succinate) under osmotic stress conditions in the presence of an exogenous osmoprotectant. The difference in growth between light-exposed and dark-maintained cells suggests that BphP2 and/or BphR are indeed functional in B728a. Our
RNAseq study was conducted in a rich medium (King’s B), a medium that does not confer a particularly high osmotic stress. The difference in media in the RNAseq study versus our growth study may be another reason why we did not see any transcriptional regulation by BphP2, as
BphP2 may be functional only under stressful conditions. Based on the growth of the ∆bphP2R mutant, BphP2 or BphR appear to mediate a growth response to osmotic stress in the dark. Many
Pseudomonas syringae strains contain orthologs to BphP2 (16) and further testing of growth under dark conditions with and without the presence of an osmotic stress in the strains with
BphP2 orthologs would provide information on BphP2-regulation of culture growth.
100
Anticipatory light-enhanced osmotolerance as a stress response strategy
We identified a phenomenon in pseudomonads that we call anticipatory light-enhanced osmotolerance, and we propose that it is a strategy employed to cope with environmental change.
Here, I would like to explore how this phenomenon in pseudomonads fits into a framework of strategies that microbes use to respond to environmental stimuli and enhance their fitness. These strategies include direct regulation, cross-protection, and symmetrical and asymmetrical adaptive prediction. These responses all aim to maintain critical functions upon experiencing a stress. In direct regulation, a cell recognizes a stressor and launches a response. In cross protection, a cell is exposed to one or more unrelated stimuli and induces multiple stress resistance mechanisms
(7; 13; 20). In adaptive prediction, a cell responds to one of two stimuli that are linked, temporally or otherwise, and this linkage allows for responses to multiple stressors (19).
Whereas stimuli for cross protection tend to be environmental stresses, such as osmotic stress, stimuli that induce adaptive prediction responses are often environmental signals that are not necessarily stressful, such as light, although some signals become stressful with an increased dosage. Similarly, whereas cross-protection responses are not always evaluated within the context of the stresses within a habitat, adaptive prediction responses are, as are their roles in microbial fitness within these habitats. Symmetrical adaptive prediction resembles cross- protection in that the stimuli are not necessarily temporally ordered, but they must be linked. In contrast, asymmetrical adaptive prediction occurs when the stimuli are consistently temporally linked, that is, the appearance of a secondary stimulus must be predictable based on the appearance of the first. Organisms that use asymmetrical adaptive prediction activate a response to the secondary stressor upon the appearance of the first, thereby increasing fitness upon exposure to the secondary stress (18; 19). Given the dependable temporal coupling of light and evaporative water loss on terrestrial surfaces such as leaves, the phenomenon of anticipatory 101 light-enhanced osmotolerance should be evaluated as an asymmetrical adaptive prediction strategy that enhances the fitness of at least some of the pseudomonads that are plant-associated.
Anticipatory light-enhanced osmotolerance may not fully satisfy the criteria for an adaptive prediction response
Of the response strategies defined in the literature, anticipatory light-enhanced osmotolerance aligns most clearly with asymmetric adaptive prediction in that the initial stimulus
(light) primes the cells against a second, temporally-linked stimulus (osmotic stress) that occurs naturally within the microbe’s habitat. However, three criteria have been specified for deeming a response adaptive prediction (19): i) the initial stimulus primes against the second stimulus, but the second stimulus does not prime against the first (i.e., the response must be asymmetric and in agreement with the temporal order of the stimuli in the environment); ii) priming by the initial stimulus imposes a fitness cost in the absence of the second stimulus (i.e., the response to the initial stimulus is only adaptive when the two stimuli are temporally coupled); and iii) priming against the second stimulus does not occur in response to other unrelated stimuli (i.e., anticipation is specific to the temporally coupled stimuli) (19).
Anticipatory light-enhanced osmotolerance does not clearly satisfy the first criterion.
Pre-exposure to light primes B728a for enhanced growth upon osmotic stress, satisfying the first half of the first criterion. However, the light wavelengths that induce enhanced osmotolerance, namely red and far-red light, are not themselves stressful (i.e., they do not cause cell damage), nor do they offer a direct benefit to the cell (i.e., they are not an energy source for the cells).
Thus, in this instance, light is solely a signal and therefore, by our reasoning, cannot be primed for or against. The environmental factors that can function solely as information (e.g., far-red light, magnetic fields) are relatively few compared to the nutrients and stresses that cells 102 encounter and could easily have been overlooked when these criteria were outlined. We advocate that the second half of the first criterion does not apply for such factors.
Regarding the second criterion, we have not yet determined if light priming imposes a fitness cost in the absence of osmotic stress. Our previous study demonstrated that swarming motility is delayed under light exposure compared to the dark (17), suggesting that light exposure can repress certain functions in B728a. Additionally, two other pseudomonads tested in this work (Pss HS191 and Pss B301D-R) exhibited less cumulative growth after light pre- exposure compared to dark-maintained cultures when challenged with a lower level of osmotic stress (0.75 M NaCl); this pattern was reversed when challenged with 1 M NaCl. This suggests that light priming can incur a fitness cost if the benefits of improved osmotolerance are not needed. We hypothesize that Pss HS191 and Pss B301D-R are inherently more osmotolerant than Pss B728a, such that light-induced upregulation of osmotolerance-related functions for Pss
HS191 and Pss B301D-R was unnecessary at 0.75 M NaCl and prompted needless reallocation of resources towards the stress response and away from cell growth and replication. Future experiments will be needed to empirically evaluate the fitness costs of light priming in the absence of osmotic stress, and at low stress levels, in Pss B728a and other pseudomonads.
Regarding the third criterion, additional experiments are also needed to determine if osmotolerance is enhanced specifically by light or also by other unrelated stimuli. Yu et al. (29) showed that osmotolerance-related genes are slightly upregulated in response to low levels of nitrogen and iron, but expression levels were substantially lower than those induced by light and whether these expression levels correlated with enhanced osmotolerance was not examined. The term adaptive prediction is sufficiently specific in its requirements as to exclude phenomena that, like light-enhance osmotolerance, are anticipatory in nature. Gaining a better understanding of 103 how organisms sense environmental cues and respond in an anticipatory manner is key to our comprehension of microbial fitness.
The integration of the BphP1 and AlgU regulon may occur via direct protein interactions in the membrane
A major goal of this work was to identify regulatory components involved in photosensing in Pss B728a. One of the genes whose expression depended on BphP1 is algU, which codes for the ECF sigma factor AlgU. AlgU has a major role in regulating the response to environmental stress and is required for the response to osmotic stress and light-enhanced osmotolerance. In the absence of envelope stress, AlgU associates with the anti-sigma factor protein, MucA, which is embedded in the cell membrane; this sequestration prevents AlgU from binding to the promoters of genes in the AlgU regulon. Stresses such as hyperosmolarity that cause envelope stress promote the misfolding of outer membrane proteins, activating a sequence of events involving interactions of MucA with other membrane and periplasmic proteins (e.g.,
MucB, MucD, MucP and AlgW) that ultimately degrade MucA and release AlgU into the cytoplasm. Many of these proteins have PDZ domains (e.g., MucD, MucP and AlgW), which indicate that they may interact as a large signal transduction complex. Based on this model of post-translational regulation of AlgU, the known autoregulation of AlgU on algU expression, and the predicted localization of BphP1 to the cytoplasmic membrane, we hypothesize that
BphP1 interacts directly with regulatory components of AlgU to integrate environmental stress response pathways. Moreover, studies in Pseudomonas aeruginosa (Pa) have identified some probable regulatory components involved, namely AlgB and KinB. In particular, the Pa bacteriophytochrome was recently found to phosphorylate AlgB (22), a transcriptional activator that is subject to dephosphorylation by KinB (6). Interestingly, although KinB likely has many 104 functions (4; 5), one function is to negatively regulate the proteolysis of MucA by AlgW (6), thus influencing the availability of the AlgU sigma factor for gene regulation.
Plant photosensory proteins often function within multiprotein complexes, as predicted for BphP1 in B728a
Many plant photosensory proteins function in complexes involving protein-protein interactions, providing precedence for our model of how BphP1 functions in pseudomonads. For example, plant phototropins interact with cryptochromes and phytochromes to regulate phototropism (11). They also directly interact with a variety of signaling proteins, including
NPH3 (non-phototropic hypocotly3), RPT2 (root phototropism2), PKS (phytochrome kinase substrate) proteins, and ABCB (ATP binding cassette B) transporters, to name a few, all which have unique roles in the downstream regulation of phototropism (9; 11; 15). Additionally, phototropins localize to various positions within the cell, from membrane to cytoplasm to Golgi apparatus to nucleus, depending on the state of light sensing (1; 11). Phototropins therefore illustrate how light sensing in plants is complicated and involves multiple regulators and regulatory mechanisms (1; 9; 11; 15). Similarly, plant phytochromes can function as kinases and regulate via phosphorylation of other proteins or other types of protein-protein interactions and post-translational modifications (14; 23). Plant phytochromes typically reside in the cytoplasm, but upon sensing light are translocated to the nucleus where they form photobodies by interacting with phytochrome interacting factors (PIFs) and phytochrome interacting factor-like (PIL) proteins, which allow for direct regulation of plant gene expression (14; 15; 23; 28). Thus, both the blue light-sensing phototropin and cryptochromes and the red/far-red light-sensing phytochromes of plants function by interacting with a number of other proteins in the cell. 105
Insights from LOV- and phytochrome-mediated signal transduction mechanisms in microorganisms
The signal transduction mechanisms by which light sensing by LOV domain containing proteins is transmitted to other proteins within the cell are largely unknown, especially in bacteria. One study conducted on Pseudomonas syringae pv. tomato (Pst) found that PstLOV is involved in negatively regulating rpoD, rpoS, rpoN, hrpL, and gacS/gacA, which all encode major regulators affecting stress responses and virulence, suggesting that PstLOV is a global regulator of gene expression (21). However, the fact that the loss of lov had relatively little phenotypic impact is not consistent with LOV regulation of these key, central regulators (2).
Another study has suggested a role for LitR (light-induced transcription regulator) proteins in
LOV-mediated regulation in Pseudomonas putida (Pp) KT2440. LitR proteins are newly studied transcriptional regulators in the MerR family (24). Pp KT2440 contains three Class II LitR homologs, which are widely conserved among Pseudomonas species (25). Two of the LitR proteins in KT2440 are encoded by genes that are adjacent to a gene that encodes PpLOV (24).
The three LitR proteins in KT2440 function as light-dependent negative regulators of a subset of
PpLOV-dependent, light-sensitive genes. These results suggest that the LitR transcriptional regulators interact with PpLOV (24). The only other documented incidence of LOV interacting with a transcription factor occurs in the fungus Neurospora crassa where a LOV protein makes up one half of the white-collar complex (WCC). Under dark conditions, WCC binds to promoter regions of light-responsive genes such as additional transcription factors, circadian clock genes, and a gene coding for another blue light-sensing LOV domain containing protein. When the
LOV domain of the WCC is activated, a conformational change is induced and transcription occurs (2). 106
The signal transduction pathways by which light sensing by phytochromes is transmitted to other proteins via their histidine kinase activity are also poorly understood. Phytochrome- mediated regulation has been characterized in a few microorganisms. For example, as mentioned above, the Pa bacteriophytochrome has been shown to phosphorylate AlgB, a transcriptional regulator that regulates biofilm formation by repressing quorum sensing and exopolysaccharide production genes (22). This is a broadly conserved regulatory relationship as PaBphP can phosphorylate AlgB from Rhodospirillum centenum, Achromobacter xylosoxidans, and
Pseudomonas putida, which are members of the α-, β-, and γ-Proteobacteria, respectively (22). In
Bradyrhizobium, a bacteriophytochrome regulates transcription of genes within a photosynthesis gene cluster, including the transcription of a regulator PpsR1 by antagonizing PpsR2, a second transcriptional regulator (12). BphP1 interacts directly with PpsR2 in another strain,
Rhodopseudomonas palustris, by forming a heterodimer thereby inactivating the repression of
RpPpsR2 (3). In Xanthomonas oryzae pv. oryzae, a bathyphytochrome that has an EAL domain rather than a histidine kinase domain modulates c-di-GMP concentrations, which influences various behaviors (26). Perhaps the best characterized signal transduction system involving a phytochrome in a non-phototrophic microbe is in the fungal species Aspergillus nidulans. Three phosphorylated targets have been identified for the phytochrome AnFphA, which has a histidine kinase domain. Phosphorylation of the transcriptional regulator SakA, an osmotic stress regulator, requires the presence of AnFphA. Similarly, phosphorylation of the proteins YpdA and
SskA requires AnFphA, however, this phosphorylation occurs in the dark, and is repressed in the light due to light sensing by AnFphA. This light-mediated repression of phosphorylation triggers transcription by the transcriptional activator AtfA of genes involved in sexual development and spore germination (30). Taken together, these results suggest a conserved role for photosensory 107 proteins interacting directly with regulatory factors to modulate cellular function and environmental responses.
Future directions in identifying photosensory-regulator interactions in Pss B728a
Bacteriophytochromes are predicted to be membrane associated, but we are currently unaware of studies examining their subcellular localization. We predict that there may be similarities in how regulation occurs between the phytochromes from plants and microorganisms. Phytochromes in plants move between two subcellular locations, the cytoplasm and the nucleus. While bacteria do not contain subcellular compartments, we hypothesize that bacteriophytochromes could co-localize with regulator proteins to initiate changes in transcription in a photobody-type manner. Specifically, we have shown that BphP1 regulates the expression of the alternative sigma factor AlgU, which is inactivated during sequestration by the membrane-bound anti-sigma factor MucA. Upon sensing light, BphP1 could antagonize the anti- sigma factor thereby allowing for the release of AlgU and the subsequent activation of downstream genes involved in the stress response. In order to test this hypothesis, we propose conducting protein-protein co-localization studies with BphP1 and known regulators of stress response pathways in Pss B728a. While co-localization studies can be performed in many ways, described in detail in a review by Dwane and Kiely (8), I am most interested in fluorescence microscopy of fluorescently-labeled proteins, as this would provide information on both co- localization of proteins with BhpP1 and cellular localization, thus clarifying whether BphP1 is in fact membrane-associated or not.
In conclusion, this thesis identified Pss BphP1 as a central component to changes in gene expression upon light sensing by Pss B728a. Moreover, it has a central role in an anticipatory response to osmotic stress and thus functions to help integrate environmental signals by triggering response pathways. This thesis also provided evidence that anticipatory light-enhanced 108 osmotolerance is conserved across some plant-associated Pseudomonas species. In the future, these findings will be useful for investigating additional light- and BphP1- regulated phenotypes and determining if light functions as an anticipatory cue to enhance responses to other temporally-linked environmental stresses.
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