The Evolutionary Diversity and Biological Function of Phenazine Metabolite Biosynthesis in Burkholderia SPP

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Master's Theses

Summer 2019

Samuel Hendry University of Southern Mississippi

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Recommended Citation Hendry, Samuel, "The Evolutionary Diversity and Biological Function of Phenazine Metabolite Biosynthesis in Burkholderia SPP" (2019). Master's Theses. 653. https://aquila.usm.edu/masters_theses/653

This Masters Thesis is brought to you for free and open access by The Aquila Digital Community. It has been accepted for inclusion in Master's Theses by an authorized administrator of The Aquila Digital Community. For more information, please contact [email protected]. THE EVOLUTIONARY DIVERSITY AND BIOLOGICAL FUNCTION OF PHENAZINE METABOLITE BIOSYNTHESIS IN BURKHOLDERIA SPP

Samuel Hendry

A Thesis Submitted to the Graduate School, the College of Arts and Sciences and the School of Biological, Environmental, and Earth Sciences at The University of Southern Mississippi in Partial Fulfillment of the Requirements for the Degree of Master of Science

Approved by:

Dmitri Mavrodi, Committee Chair Janet Donaldson Mohamed Elasri

______Dr. Dmitri Mavrodi Dr. Jake Schaefer Dr. Karen S. Coats Committee Chair Director of School Dean of the Graduate School

August 2019

Samuel Hendry

2019

Published by the Graduate School

ABSTRACT

Burkholderia encompass a group of ubiquitous Gram-negative bacteria that include numerous saprophytes, as well as several species that cause infections in animals, immunocompromised patients, and plants. Some species of Burkholderia produce colored redox-active secondary metabolites called phenazines (Phz). In the model opportunistic pathogen Pseudomonas aeruginosa, phenazines strongly contribute to the competitiveness, formation of biofilms, and virulence in multiple models of infection.

Similar depth of knowledge on the diversity, biosynthesis, and biological functions of phenazines in Burkholderia is missing. This project aimed to bridge this gap in knowledge by focusing on phenazine pathways of B. lata and closely related species. My results revealed that phz genes are present in genomes of many Burkholderia that have a worldwide origin and belong to different species of the genus. Most Phz+ strains were in the Bcc group, but the capacity to synthesize phenazines was also found in some isolates of the B. pseudomallei clade and the plant pathogen B. glumae. My findings also suggest that the phenazine biosynthetic pathway of Burkholderia has a complex evolutionary history, which likely involved horizontal gene transfers among several distantly related groups of producing organisms. I also analyzed isogenic mutants and plasmid deletion derivatives of Burkholderia lata, which helped to identify phenazines produced by species of the ubiquitous B. cepacia (Bcc) group and characterize the role of phenazine- modifying genes in the synthesis of 4,9-dihydroxyphenazine-1,6-dicarboxylic acid dimethylester. My functional studies failed to link the production of phenazines with the capacity of Burkholderia to kill fruit flies and rot onions, but other experiments revealed

a link between the presence and amount of phenazines and the dynamics of biofilm growth flow in the flow cell and static experimental systems.

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ACKNOWLEDGMENTS

I would like to express my deepest gratitude to Drs. Dmitri Mavrodi and Olga

Mavrodi. Without heir tireless work, motivation, and never-ending help, I would have never reached this point in my education. The high standards implemented in their lab allowed for me to grow into a confident and well-rounded researcher. Thanks to my committee member Dr. Mohamed Elasri for teaching me and sharing his equipment and wealth of knowledge in the realm of biofilm studies. Thanks also to my committee member Dr. Janet Donaldson for her help and encouragement, as well as teaching me much in bacteriology. I also thank Dr. Wulf Blankenfeldt and Stephan Steinke from the

Helmholtz Center for Infection Research in Germany for their assistance in extracting and performing analytical analyses on phenazine metabolites. I could not have done this without my supportive and loving wife, Mary Katherine, and our dog Luna. Many thanks to my Mavrodi lab family for all their help.

This work was supported by startup funds in Dr. Mavrodi’s lab, the EAGLE

Scholarship Program for Undergraduate Research, and by Mississippi INBRE, funded by an Institutional Development Award (IDeA) from the National Institute of General

Medical Sciences of the National Institutes of Health under grant number P20GM103476.

TABLE OF CONTENTS

ABSTRACT ...... ii

ACKNOWLEDGMENTS ...... iv

LIST OF TABLES ...... vii

LIST OF ILLUSTRATIONS ...... viii

LIST OF ABBREVIATIONS ...... ix

CHAPTER I – INTRODUCTION ...... 1

1.1 Burkholderia spp. are diverse and economically important microorganisms...... 1

1.2 The diversity and biosynthesis of microbial phenazines ...... 5

1.3 Biological function of phenazine metabolites...... 8

1.4 Phenazine production in Burkholderia spp...... 11

1.5 Aims of this study ...... 12

CHAPTER II - MATERIALS AND METHODS ...... 14

2.1 Screening the newly-sequenced and publicly-available genome sequences of

Burkholderia for the presence of phenazine biosynthesis genes...... 14

2.2 Genetic analysis of phenazine biosynthesis in Burkholderia spp...... 16

2.3 Pathogenicity assays with the fruit fly Drosophila melanogaster and onions ...... 17

2.4 Flow cell and static biofilm assays ...... 18

CHAPTER III – RESULTS ...... 21

3.1 Screening Burkholderia genomes for the presence of phenazine genes ...... 21

3.2 Genetic analysis of phenazine biosynthesis in B. lata ATCC 17760 ...... 27

3.3 Biological function of phenazine compounds in B. lata ATCC 17760 ...... 30

CHAPTER IV – DISCUSSION...... 38

REFERENCES ...... 43

LIST OF TABLES

Table 3.1. Dose-response assays with B. cepacia ATCC 25416 and B. lata ATCC 17760

...... 32

Table 3.2. Fruit fly pathogenicity assays with B. lata ATCC 17760 and its isogenic phenazine mutant derivatives ...... 33

Table 3.3. Onion tissue maceration assays with different Burkholderia spp ...... 34

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LIST OF ILLUSTRATIONS

Figure 1.1 Phenazine metabolites and biosynthesis ...... 7

Figure 3.1 16s rRNA phylogeny of Burkholderia spp...... 22

Figure 3.2 Multi-locus sequence typing phylogeny of Burkholderia spp...... 23

Figure 3.3 Contrasting phylogenies using phz proteins and housekeeping enzymes ...... 24

Figure 3.4 Gene organization of various Burkholderia phz loci ...... 25

Figure 3.5 Flanking regions surrounding the phz loci ...... 27

Figure 3.6 Phenazine intermediates identified from culture of B. lata ATCC 17760 ...... 29

Figure 3.7 Genetic organization of phz gene cluster in B. lata ATCC 17760 ...... 30

Figure 3.8 Survival curves for D. melanogaster infected with Burkholderia spp...... 31

Figure 3.9 Onion tissue maceration by B. cepacia ATCC 25416 ...... 34

Figure 3.10 Graph of % coverage of biofilm in BioFlux microfluidistic system ...... 36

Figure 3.11 Static biofilm formation ...... 37

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LIST OF ABBREVIATIONS

AOCHC 6-amino-5-oxocyclohex-2-ene-1-carboxylic acid

Bcc Burkholderia cepacia complex

DHHA trans-2,3-dihydro-3-hydroxyanthranilic acid

KMB King’s Medium B Broth

PCA Phenazine-1-carboxylic acid

PDC Phenazine-1,6-dicarboxylic acid

Phz Phenazine

TSB Tryptic Soy Broth

CHAPTER I – INTRODUCTION

1.1 Burkholderia spp. are diverse and economically important microorganisms

Burkholderia is a ubiquitous group of Gram-negative bacteria that contain numerous saprophytes, as well as species that are associated with infectious diseases, hospital-acquired infections and necrotizing pneumonia in individuals with cystic fibrosis

(Woods & Sokol, 2006). These organisms were previously classified as Pseudomonas, but the advent of molecular and genetic techniques ultimately identified Burkholderia as members of a distinct genus within Betaproteobacteria (Yabuuchi et al., 1992; Estrada-de

Los Santos et al., 2015; Eberl & Vandamme, 2016). The taxonomic status of these microorganisms was revised further in 2014 by dividing the group into two genera, with

Burkholderia containing opportunistic pathogens of animal and plants, and a new genus

Paraburkholderia harboring environmental species and nitrogen-fixing mutualists

(Sawana, Adeolu, & Gupta, 2014). Subsequent studies established the polyphyletic nature of the Burkholderia and Paraburkholderia groups and separated several species into a new genus, Caballeronia (Dobritsa & Samadpour, 2016). There is an ongoing discussion about further revisions of the genus Paraburkholderia and advancing the taxonomic status of some species to the level of new genera.

The genus Burkholderia encompasses over 100 species that include several economically important pathogens of humans, animals, and plants (Estrada-de Los Santos et al., 2015). Three distinct lineages are recognized within the Burkholderia sensu stricto group: 1) the B. pseudomallei clade of mammalian pathogens, 2) the Burkholderia cepacia complex (Bcc) of opportunistic pathogens, and 3) the B. glumae/B. gladioli clade of plant pathogens (Sawana, Adeolu, & Gupta, 2014). The B. pseudomallei clade consists

of closely related infectious agents that are associated with serious infections in humans and animals (Wiersinga et al., 2006; Whitlock, Estes, & Torres, 2007; Losada et al.,

2010). Burkholderia pseudomallei is a common soil and freshwater saprophyte, but under certain conditions, can infect individuals with underlying risk factors such as diabetes, chronic renal impairment, congestive heart failure, corticosteroid therapy, and malignancy (Foong, Tan, & Bradbury, 2014). This organism is a causative agent of melioidosis, a condition associated with nearly 20% cases of acquired septicemia in

Southeast Asia and Northern Australia. During melioidosis, the pathogen often attacks lungs causing a chronic pulmonary infection but later disseminates systemically through the bloodstream causing life-threatening septicemia (Dance, 1991). Burkholderia pseudomallei is capable of intracellular motility, has multiple virulence factors (i.e., surface polysaccharides, type III and type VI protein secretion systems), forms biofilms and is intrinsically resistant to commonly used antibiotics making the successful treatment of melioidosis a challenge (Stone et al., 2014; Sawasdidoln et al., 2010). B. mallei has been traditionally considered a distinct species in the genus Burkholderia, but with the advent of the multi-locus sequence typing was re-classified as a subspecies of B. pseudomallei (Godoy et al., 2003). Unlike typical B. pseudomallei, this organism is an obligate mammalian pathogen with a smaller multi-replicon genome. Although primarily a pathogen of equines, in which B. mallei causes glanders, the bacterium is also capable of infecting humans through contact with diseased horses, mules, and donkeys (Van

Zandt, Greer, & Gelhaus, 2013). The pathogen is transmitted by inhalation, via mucous membranes and cuts in the skin, and causes both chronic and acute infections accompanied by fever, pustules, abscesses, and pneumonia. If left untreated, the fatality

rate of B. mallei infections is as high as 95%. Due to the highly virulent nature of B. mallei among equine, and the ease to distribute in an aerosol form, it was used in World

War I as biological warfare to infect the opposing force’s horses and livestock (Godoy et al., 2003; Wheelis, 1998). The United States Centers for Disease Control and Prevention

(CDC) list B. pseudomallei and B. mallei among the category B priority pathogens and potential bioterror agents.

The Burkholderia cepacia complex (Bcc) is named after the type species of the genus and includes microorganisms that are common in the environment, though under favorable conditions can act as opportunistic human pathogens (Sousa et al., 2011).

Many Burkholderia spp. are typical saprophytes and particularly abundant in areas with acidic soils (Stopnisek et al., 2014), while others colonize eukaryotic hosts and include both commensals and economically important pathogens of plants and animals. Members of the Bcc group also include endosymbionts of insect that increase their hosts' fitness by protecting them from pathogens and parasites (Kim et al., 2015; Lee et al., 2017), as well as species that establish close associations with fungi, including arbuscular mycorrhizae, and benefit from the utilization of nutrients present in fungal exudates (Stopnisek et al.,

2016). Species of the Bcc complex are also well-known for the ability to catabolize different organic compounds as energy sources, produce an impressive array of secondary metabolites, and their plant growth promoting and biocontrol properties (Eberl

& Vandamme, 2016). However, despite the tremendous biotechnological potential, the broader acceptance of Bcc organisms for the control of plant diseases and remediation of sites contaminated with recalcitrant pollutants has been hampered by the inability to discriminate between the beneficial environmental and the clinical strains.

Over the past few decades, many members of the Bcc group have emerged as causative agents of severe nosocomial infections in immunocompromised patients and individuals with cystic fibrosis (Sousa et al., 2011; Mahenthiralingam et al., 2005).

Various strains of the B. cepacia complex have also been associated with bacteremia in patients with no underlying disease and identified as the cause of hospital-acquired sepsis in patients (Kim et al., 2016). These organisms harbor an impressive array of virulence determinants (i.e., type III, IV, and VI protein secretion systems, adhesins, exoproteases, and a lipase), some of which are regulated by homoserine lactone-producing quorum- sensing (Loutet & Valvano, 2010). Members of the B. cepacia group also are intrinsically resistant to a wide range of antibiotics, form robust biofilms, and have multiple mechanisms to resist oxidative stress and sequester iron, making Bcc infections particularly challenging to treat (Sawasdidoln et al., 2010; Lewis & Torres, 2016).

Finally, species of the B. glumae/B. gladioli clade live in association with different plants and colonize plant tissues externally or endophytically (Compant et al.,

2008). Many species of this group maintain commensal relationships with their hosts, but some act as plant pathogens. For example, B. gladioli pv. allicola causes the slippery skin of onions, during which the pathogen affects both the aboveground parts of the plant and the bulbs (Stoyanova et al., 2007). In early stages of the disease the bulbs appear healthy, but later the infected tissues become water-soaked and eventually decay. Interestingly, a similar condition known as the sour skin of onion is caused by many strains of B. cepacia, which can infect plants in the field or at storage and lead to the development of a soft, watery rot of bulbs (Stoyanova et al., 2007). Other pathovars of B. gladioli damage bulbs of gladioluses, irises, infect certain species of orchids (Keith, Sewake, & Zee,

2005), while B. gladioli pv. agaricicola produces a variety of antifungal secondary metabolites and is an important pathogen in the mushroom industry, where it causes a soft rot of button mushrooms (Agaricus bisporus) (Elshafie et al., 2016). Another serious emerging phytopathogen in this group is B. glumae, which is associated with the bacterial blight of rice. This disease was first reported in the 1950s in Japan but later spread to other parts of Asia, South and Central America and the United States (Ham, Melanson, &

Rush, 2011). B. glumae secretes several phytotoxins, extracellular degradative enzymes, and type III secretion effectors causing in rice floret sterility, sheath rot, inhibition of seed germination, and seedling blight (Angus et al., 2014; Naughton et al., 2016). Although B. glumae and B. gladioli are typically considered plant pathogens, strains of both species have been isolated from immunocompromised individuals or patients with cystic fibrosis further highlighting the remarkable versatility of these organisms and their capacity to adapt and thrive in diverse environments (Devescovi et al., 2007; Martinucci et al.,

2016).

1.2 The diversity and biosynthesis of microbial phenazines

My project focused on strains of Burkholderia that produce the colored, redox- active secondary metabolites called phenazines (Phz). Synthesis of phenazines is most common among Actinobacteria, especially Streptomyces spp., but it also occurs within two clades of Gram-negative bacteria (Mavrodi et al., 2006). Fluorescent Pseudomonas spp. are -Proteobacteria and the best-characterized phenazine producers, with strains of

P. fluorescens, P. aureofaciens (now classified as P. chlororaphis) and P. aeruginosa known to synthesize these compounds. Another Phz+ gamma proteobacterium, Pantoea agglomerans Eh1087, belongs to the Enterobacteriaceae and synthesizes the phenazine

derivative D-alanylgriseoluteic acid (Giddens, Houliston, & Mahanty, 2003). Among β-

Proteobacteria, phenazines are produced by Burkholderia spp. Apart from different

Eubacteria, the only other known phenazine producers are some archaea that employ membrane-embedded phenazines as electron carriers in methanogenesis (Abken et al.,

1998).

Studies in the early 1970s in which radiolabeled compounds were added to intact bacterial cells identified phenazines as derivatives of the shikimic acid pathway

(reviewed by Turner & Messenger, 1986). These experiments concluded that the synthesis of phenazines begins with chorismic acid, and that the phenazine tricycle is formed by the condensation of two chorismate molecules with glutamine used as the source of nitrogen in the heterocyclic nucleus. Phenazine-1,6-dicarboxylic acid (PDC) was proposed as the first phenazine formed, although its incorporation into the phenazine core could not be detected in some bacteria. Further progress towards understanding the reactions leading to formation of the phenazine scaffold remained obscure until the

1990s, when phenazine biosynthesis genes were cloned and sequenced from several

Pseudomonas species (Pierson et al., 1995; Mavrodi et al., 1998; Mavrodi et al., 2001).

A conserved seven-gene operon, phzABCDEFG, present in two copies in P. aeruginosa

(Mavrodi et al., 2001), was implicated in the synthesis of PCA. I refer to these genes as the “core phenazine biosynthesis genes”.

Microbial phenazines are structurally diverse but all share a conserved nitrogen- containing tricyclic core (Figure 1.1), which is assembled by products of the core biosynthesis genes. The synthesis of the phenazine tricycle starts with the anthranilate- synthase-like enzyme PhzE and the isochorismatase PhzD together catalyzing the

conversion of the chorismic acid to trans-2,3-dihydro-3-hydroxyanthranilic acid

(DHHA), which is then isomerized to 6-amino-5-oxocyclohex-2-ene-1-carboxylic acid

(AOCHC) by PhzF (Figure 1.1) (Mentel et al., 2009).

Pyocyanin Phenazine-1-carboxylic Phenazine-1,6- Endophenazine A acid (PCA) dicarboxylic acid (PDC)

D-Alanyl-griseoluteic acid Saphenamycin

PhzE Chorismic acid ADIC PhzD

PhzA/B G PhzF DHHA

HHPDC AOCHC1

PhzG AOCHC2

PCA PDC

Figure 1.1 Phenazine metabolites and biosynthesis

(A) Structures of some phenazine compounds produced by Pseudomonas, Streptomyces, and Enterobacter, and (B) biosynthesis of the common phenazine precursors PCA and PDC from chorismic acid by products of highly conserved core Phz genes. ADIC, - 2-amino-

2-desoxyisochorismic acid; DHHA - trans-2,3-dihydro-3-hydroxyanthranilic acid; AOCHC - 6-amino-5-oxo-2-cyclohexene-1- carboxylic acid; HHPDC - hexahydrophenazine-1,6-dicarboxylic acid; PCA - phenazine-1-carboxylic acid; PDC – phenazine-1,6- dicarboxylic acid. 7

Next, two AOCHC molecules are fused into a tricycle intermediate via the head- to-tail condensation catalyzed by the small ketosteroid isomerase-like protein PhzA/B.

The final step of the biosynthesis involves the flavin-dependent oxidation that is catalyzed by the FMN-dependent oxidase PhzG and yields phenazine-1-carboxylic acid

(PCA) in Pseudomonas (Blankenfeldt & Parsons, 2014). In other groups of Phz+ bacteria, this step results in the formation of phenazine-1,6-dicarboxylic acid (PDC). The last core enzyme, PhzC, is a 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase that acts upstream of phenazine biosynthesis to ensure an abundant flow of metabolites into the shikimate pathway. In addition to highly conserved core genes, most producers carry other phenazine genes that vary in number and biological function depending on the species of Phz+ bacteria. These auxiliary genes encode enzymes that covert PCA and

PDC into species-specific phenazine metabolites such as those shown in Figure 1.1, as well as proteins that perform efflux, resistance, and regulatory functions (Mavrodi et al.,

2010).

1.3 Biological function of phenazine metabolites

Natural phenazines were originally described as microbial pigments with unidentified biological function. However, studies conducted over the past two decades have revealed that these metabolites play an important role in the biology of the producing bacteria. Most phenazines are broadly inhibitory to bacteria, fungi, and parasites, and some have antitumor activity (Smirnov & Kiprianova, 1990). Structurally simple phenazines are produced by beneficial strains of the Pseudomonas fluorescens complex and P. chlororaphis on the roots of plants, where they suppress soilborne diseases caused by fungal pathogens (Chin-A-Woeng, Bloemberg, & Lugtenberg, 2003).

D-alanylgriseoluteic acid, produced by Pantoea agglomerans on apple flowers, contributes to the suppression of Erwinia amylovora, which causes fireblight disease of apples and pears (Giddens, Houliston, & Mahanty, 2003). The antibiotic activity of pyocyanin, a blue phenazine synthesized by Pseudomonas aeruginosa, was known as early as in the late 1850s (Fordos, 1860; Gessard, 1882), but more recent studies demonstrated that this metabolite enables the producing strains to kill Drosophila melanogaster and Caenorhabditis elegans (Mahajan-Miklos et al., 1999; Lau et al.,

2003; Cezairliyan, et al., 2013). Pyocyanin is critical for lung infection by P. aeruginosa in mice (Lau et al., 2004), and has been detected at concentrations up to 10-4 M in the sputa of patients with cystic fibrosis (Mavrodi, Blankenfeldt, & Thomashow, 2006).

These and other results suggest that pyocyanin is a host-nonspecific pathogenicity factor.

In contrast, some phenazines produced by Streptomyces spp. are not cytotoxic in eukaryotes and hold promise as anticancer or anti-infective drugs (Laursen & Nielsen,

2004).

Much of the biological activity of phenazines is due directly to their redox properties (Hassan & Fridovich, 1980; Hassett et al., 1992). Pyocyanin can undergo cellular redox-cycling in the presence of oxygen and reducing agents including NADH

¯• and NADPH, causing the accumulation of toxic superoxide (O2 ) and hydrogen peroxide

(H2O2) (Hassan & Fridovich, 1980). This also raises questions as to how phenazine producers protect themselves from phenazine toxicity. In E. coli, two redox-sensing global regulators, SoxR and OxyR, control stress responses to superoxide and hydrogen peroxide, respectively. P. aeruginosa encodes homologs of both regulators, and while the global role of OxyR appears to be preserved, the function of SoxR is reduced exclusively

to the control of a small group of genes induced by pyocyanin or the redox-active compound paraquat (Dietrich et al., 2008). Most strongly induced by these compounds are genes encoding a putative flavin-dependent monooxygenase and components of a proton-driven pump, MexGHI-OpmD, that belongs to the resistance-nodulation-division

(RND) superfamily and is involved in active efflux of pyocyanin (Dietrich et al., 2006).

Among other mechanisms contributing to PYO resistance in P. aeruginosa are high levels of superoxide dismutase and catalase activity (Hassett et al., 1992).

Though traditionally considered secondary metabolites of no direct benefit to the cells that produce them, phenazines synthesized by bacteria in their native habitats clearly contribute to competitiveness and long-term survival. Thus, phenazine-producing pseudomonads are more competitive and survive longer on the roots of wheat than their phenazine-nonproducing mutants (Mazzola et al., 1992), and pyocyanin-deficient mutants are less virulent and less competitive than wild-type P. aeruginosa in mouse acute and chronic pneumonia infection models (Lau et al., 2004). This phenomenon has been attributed to the ability of phenazines to transfer electrons to extracellular substrates,

(also known as extracellular respiration) and thus contribute to redox homeostasis in the species that produce them. Pyocyanin helps to maintain the intracellular redox balance in

P. aeruginosa by accepting electrons from NADH when other oxidants for respiration have become limited, and by stimulating the excretion, and subsequent fermentation

(bypassing oxidative phosphorylation), of pyruvate during the stationary phase (Price-

Whelan, Dietrich, & Newman, 2007). The reduction of extracellular pyocyanin could be particularly beneficial under conditions of oxygen limitation such as those found in the biofilms inhabited by P. aeruginosa in chronically-infected host tissues. Consistent with

this model, phenazines have been implicated in the anaerobic survival of P. aeruginosa

(Wang et al., 2010), and Phz- mutants of P. chlororaphis 30-84 were impaired in the formation of biofilms in a flow cell system (Maddula et al., 2006).

Phenazines can also act as electron shuttles to promote microbial mineral reduction, and it has been suggested that they could make ferric iron oxides and other nutrients in soil more accessible to microorganisms (Price-Whelan, Dietrich, & Newman,

2007). In microbial fuel cells, which actually select for phenazine-producing strains, electron shuttling due to pyocyanin produced by P. aeruginosa markedly stimulated growth and power output not only by the producer strain but also by other bacteria in mixed communities (Rabaey et al., 2004). Phenazine-1-carboxylic acid produced by P. aeruginosa makes iron more bioavailable by reducing Fe(III) to Fe(II), and, together with siderophores and heme uptake systems, facilitates the formation of biofilms in iron- limited environments, such as lungs of CF patients (Wang et al., 2011).

1.4 Phenazine production in Burkholderia spp.

The production of phenazines in Burkholderia was first reported in the 1950s-

1970s, when several studies independently reported the isolation of these secondary metabolites from strains of Pseudomonas multivorans, Pseudomonas cepacia, and

Pseudomonas phenazinium (all organisms were later reclassified as members of

Burkholderia and Paraburkholderia) (Morris & Roberts, 1959; Ballard et al., 1970; Bell

& Turner, 1973; Korth et al., 1978). Phenazines produced by B. glumae were described in a short report published much later (Han et al., 2014). The common theme of all the studies mentioned above is that they focused solely on the solvent extraction and fractionation of phenazine pigments from wild type strains and their characterization by

analytical methods. Several older studies also used isotope probing techniques to unravel biochemical details of phenazine biosynthesis (Byng & Turner, 1976; Byng & Turner,

1977; Korth et al., 1978), but the genetic basis for the production of these versatile metabolites in Burkholderia spp. remains unexplored. Several years ago, a study by

Mavrodi et al. (2010) designed primers and probes targeting the core phenazine biosynthesis gene phzF and then applied them to determine the distribution of phz genes among environmental and clinical isolates representing five different genera of Gram- positive and Gram-negative bacteria. Although the authors included in that project several phenazine-producing strains of Burkholderia, only two genome sequences of

Phz+ strains were available for the analysis, which severely limited the scope of the study. Hence, the broader picture of the distribution, organization, and evolution of phenazine pathways in Burkholderia spp. and closely related taxa remains poorly understood. Finally, it is clear that phenazines of Burkholderia have broad-spectrum antimicrobial properties and, like their counterparts from pseudomonads, can arrest the growth of numerous species of bacteria and fungi (Smirnov & Kiprianova, 1990;

Cartwright et al., 1995; Han et al., 2014). However, the broader understanding of the contribution of phenazine metabolites to the biology of saprophytic and pathogenic members of the genus Burkholderia remains unexplored.

1.5 Aims of this study

The evolution of phenazine biosynthetic genes in Burkholderia is not well studied, though phenazines were first discovered over 150 years ago. Most of our recent understanding of the diverse biological functions of phenazines and the effects they exert on higher organisms is based on a single model – the opportunistic human pathogen P.

aeruginosa. Similar information for other groups of phenazine-producing bacteria is missing. My work attempted to address this critical gap in our knowledge by focusing on phenazines produced by Burkholderia spp. These organisms are ubiquitous in the environment and contain non-pathogenic species, as well as species that cause hospital- acquired infections and colonize the lungs of individuals with cystic fibrosis. I aimed to characterize the diversity, function and biological role of phenazine pathways in B. lata

ATCC 17760, which belongs to the economically-important Burkholderia cepacia complex (Bcc). The specific objectives of my research project were:

1) To analyze the distribution, organization and evolution of phenazine biosynthesis

pathways in Burkholderia spp.

2) To conduct genetic analysis of the phenazine biosynthesis in B. lata ATCC 17760.

3) To characterize biological function of phenazines in B. lata ATCC 17760.

CHAPTER II - MATERIALS AND METHODS

2.1 Screening the newly-sequenced and publicly-available genome sequences of

Burkholderia for the presence of phenazine biosynthesis genes.

Genome sequences of phenazine-producing Burkholderia used in this study were acquired from several sources. Implementation of blast searches were used to sift through publicly-available repositories of bacteria genomes such as the National Center for

Biotechnology Information (NCBI) GenBank (Benson et al., 2014), the Joint Genome

Institute (JGI) Integrated Microbial Genomes and Microbiomes database (IGM/M)

(Markowitz et al., 2012), and the Burkholderia Genome Database (Winsor et al., 2008).

Screening was accomplished by using the tblastn algorithm (searching translated nucleotide databases using a protein query) with a cut-off E-value < 1e-5. It has previously been described that in phenazine-producing bacteria a small dimeric protein

PhzA/B catalyzes the symmetrical head-to-tail condensation of two precursor molecules and is critical for the formation of the tricyclic phenazine ring (Blankenfeldt & Parsons,

2014). The amino acid sequence of PhzA/B from B. lata ATCC 17760 was used as a query for searching databases and identifying Burkholderia genomes that harbor a phenazine loci. In addition to the publicly available genomes, draft genomes of three strains of phenazine-producing Burkholderia were also sequenced, annotated by the

Mavrodi research lab and used in bioinformatics analyses.

Draft genomes were assembled using MiSeq instrument (Illumina, San Diego,

CA) by the AgriLife Genomics and Bioinformatics Service at Texas A&M University

(College Station, TX). Raw reads were filtered using a FastQC toolkit (Andrews, 2010) followed by an assembly with the combination of BayesHammer (Nikolenko et al., 2013)

and SPAdes (Bankevich et al., 2012) implemented in the Pathosystems Resource

Integration Center (PATRIC) (Wattam et al., 2014). Scaffolds of the final assemblies that contained phenazine biosynthesis genes were extracted from genome sequences and trimmed using Geneious 10.2.3 (Biomatters, Auckland, New Zealand).

Analysis of the diversity and arrangement of the core, modifying, and auxiliary genes in phz clusters of different Burkholderia spp. was conducted in Geneious 10.2.3 using reciprocal BLAST searches and the dotplot sequence comparisons with EMBOSS dotmatcher (Rice et al., 2000). The screening of flanking regions of phenazine loci were analyzed using the DNA G+C content. Sequences were screened using tRNAscan-SE

(Schattner et al., 2005), ISfinder (Kichenaradja et al., 2010), and Island Viewer (Dhillon et al., 2015) to identify genomic islands and genes encoding tRNAs, site-specific recombinases, and transposases. Phenazine gene clusters and flanking genomic regions were aligned in Geneious using Multiple Alignment using Fast Fourier Transform

(MAFFT) 7.309 (Katoh & Standley, 2013).

The evolution of the phz pathway in Burkholderia was analyzed by establishing phylogenies inferred from sequences of core phenazine biosynthesis and housekeeping genes. Sequences were concatenated and aligned using MUSCLE (Edgar, 2004), and phylogenetic trees were inferred with Geneious Tree Builder using the neighbor joining

(NJ) algorithm. The DNA and protein distances were corrected, respectively, by

Kimura’s two-parameter (Kimura, 1980) and Jukes-Cantor (Jukes & Cantor, 1969) models of evolution.

2.2 Genetic analysis of phenazine biosynthesis in Burkholderia spp.

Amino acid sequences of PhzA, PhzF, and PhzG (also hypothesized to be involved in the formation of PCA or PDC) from B. lata ATCC 17760 were used as blastp queries to interrogate the NCBI GenBank and JGI Integrated Microbial Genomes and

Microbiomes (IMG/M) databases and the resultant hits were used to compare PhzA,

PhzF, and PhzG of Burkholderia to their counterparts from taxa that are known to produce PCA (Pseudomonas and some Streptomyces) or PDC (Burkholderia and some

Actinobacteria). Comparisons were performed using phylogenetic tools previously outlined in section 2.1. The homologs of phenazine modifying enzymes encoded by pcm1, pcm2, and pcm3 were determined using blast searches against GenBank and

IMG/M databases. The composition, physical properties, and possible cellular localization was identified using tools implemented in the European Molecular Biology

Open Software Suite (EMBOSS) (Rice et al., 2000). Functional motifs were then searched using regular expressions, generalized profiles, and hidden Markov models implemented in the MyHits database (http://myhits.isb-sib.ch/), and protein folds were predicted using the Phyre2 server (Kelley et al., 2015).

Plasmid deletion derivatives of the phenazine cluster from B. lata ATCC 17760 were used to identify phenazine products when certain biosynthetic genes were inactive or deleted. The plasmids were generated by amplifying sets of phz genes using PCR with high-fidelity KOD Hot Start DNA polymerase (Millipore Sigma) and assembling them in the cloning vector pBluescript II KS (+) (Stratagene). The cloned amplicons were single- pass sequenced to ensure the absence of unwanted mutations and sub-cloned into broad host range plasmid vectors pUCP26 (West et al., 1994) and pBBR1MCS (Kovach et al.,

1994) under the control of lac promoter. The pUCP26-based plasmids were introduced for heterologous expression into the phenazine-deficient mutant strain Pseudomonas fluorescens 2-79Z (Khan et al., 2005), while the pBBR1MCS-based constructs were used in experiments with B. lata ATCC 17760. In addition, my experiments employed phenazine-nonproducing isogenic mutants of ATCC 17760 that were constructed by interrupting genes phzA, pcm1, and pcm3 with the insertion of the p34S-Tp trimethoprim- resistance cassette (Dennis & Zylstra, 1998). The 2-79Z and ATCC 17760 derivatives carrying appropriate plasmids were generated by electroporation, selected by plating on

LB agar supplemented with tetracycline or trimethoprim, and confirmed by PCR with plasmid-specific primers. The cultures were shipped to the Dr. Wulf Blankenfeldt’s lab at the Helmholtz Centre for Infection Research in Braunschweig, Germany, where phenazine intermediates produced by plasmid deletion derivatives and isogenic mutants were extracted and analyzed by HPLC-coupled mass-spectrometry and NMR.

2.3 Pathogenicity assays with the fruit fly Drosophila melanogaster and onions

Using an assay of Castonguay-Vanier et al., I evaluated the contribution of phenazine metabolites to the virulence of Burkholderia (Castonguay-Vanier et al., 2010), by using the fruit fly Drosophila melanogaster as a surrogate host for pathogenic members of the Bcc group. Briefly, bacterial cultures were grown in Tryptic Soy Broth to an optical density (OD600) of 2. Cells were then washed with sterile 10 mM MgSO4, suspended in the same buffer, and adjusted by serial dilution to the desired concentration.

Ampicillin (500 mg/mL) was added to the cell suspensions to prevent possible infection with bacteria present on the surface of the fly. Adult flies of 8 ± 2 days were anesthetized with CO2 and pricked in the dorsal thorax using a 27-gauge syringe needle previously

dipped in the appropriate bacterial cell suspension. Control flies were pricked with a needle dipped into a solution of 10 mM MgSO4 supplemented with ampicillin. The inoculated flies were maintained at 25°C and scored daily for survival. The infection assays were performed with a minimum of 30 flies for each strain, and all experiments were repeated in multiple repetitions.

The impact of phenazines on tissue infection in onions was modeled using methods from Jacobs et al. (2008). Onions were quartered using a sharp, sterile blade, and the quartered sections were separated into individual layers. Overnight cultures of

Burkholderia spp. grown in TSB were adjusted to 107 CFU/mL, and 5-mL aliquots of bacterial suspensions were injected into onion slices using a sterile 10 mL pipette tip. The inoculated slices were incubated at 30°C on water-saturated filter paper placed in Petri plates. The degree of tissue maceration was scored after 40 hrs on a scale of 0-3 (0, no visible maceration; 1, < 33% maceration; 2, 34-65% maceration; 3, 66-100% maceration). B. cepacia ATCC 25416 is an onion pathogen and was used as a positive control. The negative control was inoculated with 5 ml of TSB. The experiment was repeated twice with five replicates of each treatment.

2.4 Flow cell and static biofilm assays

The effect of phenazines on biofilm formation was examined in B. lata ATCC

17760 and its phenazine-deficient isogenic mutant B. lata phzA and overproducing derivative carrying the pBBR1-MCS-all plasmid. The bacteria were cultured overnight at

27°C on one-third-strength King’s Medium B (1/3 KMB) plates, then scraped off the surface of the agar and suspended in 1/3 KMB broth at a density of approximately 108

CFU/mL. The standardized bacterial suspensions were inoculated into a 24-well two

inlets-one outlet flow cell plate, was connected to a BioFlux 200 microfluidistic system

(Fluxion Biosciences). The inoculum was first pumped into the flow cell at 1.0 dyn/cm2 for 6 sec, and bacteria were then allowed to attach to the channel surface for 1 h at 27°C.

Initially, a flow rate of 0.16 dyn/cm2 for the first 12-14 h, and after that, a flow rate of

0.44 dyn/cm2 was set for 36 h to continuously pump fresh 1/3 KMB through channels containing bacteria. The development of biofilms was monitored by collecting bright- field images every 20 min with an LS620 digital microscope (Etaluma). The acquired images were merged into a time-lapse movie using BioFlux software (Fluxion

Biosciences). The images were also normalized using the Threshold and Slider tools of the BioFlux Montage software (Fluxion) and used to quantify the biofilm growth by calculating total percentage of area covered by the biofilm and plotting it against the time frames.

The ability of B. lata ATCC 17760 and its mutants to form static biofilms was determined using the crystal violet biofilm assay developed by O’Toole (2011). Briefly, cultures were grown on LB plates supplemented with appropriate antibiotics for 24 h, bacteria were scraped off the agar, and OD600 was adjusted to 0.1. The normalized suspensions were diluted 1:100 in KMB broth and 100 µL aliquots were dispensed into

96-well U-bottom PVC microplates (Costar). The inoculated microplates were incubated for 48 h at 27°C, after which all wells were gently rinsed with water to remove the unattached cells and media components. Crystal violet (0.1%) was added to each well and after 15 min of staining the microplates were rinsed, dried, and the retained dye was solubilized with 30% acetic acid. The amount of the retained dye was quantified by measuring absorbance at 550 nm. The experiments were repeated four times with 60

replicates per strain, and differences between treatments were assessed by Kruskal-Wallis rank test followed by Dunn’s multiple comparisons test (P < 0.05).

CHAPTER III – RESULTS

3.1 Screening Burkholderia genomes for the presence of phenazine genes

Screening of publicly accessible databases yielded multiple genome sequences of

Burkholderia capable of producing phenazines. Overall, phenazine gene clusters were identified in 20 strains of Burkholderia and in one strain of Paraburkholderia that have a worldwide origin and belong to different species of the two genera. According to

GenBank records, most of these genomes came from strains isolated from samples of soil collected in the Northern Territory of Australia and were sequenced during a single project by the Northern Arizona University. Two more Australian strains (BDU5 and

BDU6) were isolated from soil on Badu Island located in the Torres Strait, and another one (TSV 85) from water collected in Townsville, Queensland. Both genomes of B. glumae (BGR1 and LMG 2196) belonged to phenazine-producing strains isolated from rice in Japan (Urakami et al., 1994; Jeong et al., 2003), whereas B. lata ATCC 17760 originated from soil collected in Trinidad (Stanier, Palleroni, & Doudoroff, 1966). In addition to publicly available genomes, we sequenced and annotated draft genome sequences of three strains of Phz+ Burkholderia. Two of these strains, Burkholderia sp.

2424 and PC17, were isolated in Japan from the rhizosphere of Welsh onion (Seo &

Tsuchiya, 2004). The third strain, Burkholderia sp. 5.5B, is a soil isolate and biological control agent from USA (Cartwright et al., 1995). Finally, in the Paraburkholderia group, phenazine genes were present in just one strain, the soil isolate P. phenazinium

LMG 2247 (Bell & Turner, 1973). Phylograms inferred from 16S rRNA sequences of

Phz+ strains and type strains revealed that most phenazine-producing Burkholderia were members of the Bcc group (Figure 3.1). The capacity to synthesize phenazines was also

Figure 3.1 16s rRNA phylogeny of Burkholderia spp.

Distribution of phenazine biosynthesis genes among different lineages of Burkholderia, Paraburkholderia, and Caballeronia. The neighbor-joining phylogeny was established based on 16S rRNA sequences, with Ralstonia solanacearum LMG 2299T used as an outgroup. Confirmed phenazine-producing strains are highlighted in bold font. Sequences were aligned in Geneious 10.2.3 with

MAFFT 7.309 (37). Indels were ignored, and the final alignment used for analysis contained 1,314 positions. Evolutionary distances were estimated using the Kimura two-parameter model of nucleotide substitution. Gray circles on the tree nodes indicate bootstrap values that vary between 50.0% (smallest circle) and 100% (largest circles). The final tree was visualized in iTOL (43). Values in brackets indicate GenBank accession numbers.

present in the plant pathogen B. glumae, and some strains of the B. pseudomallei clade.

Although the phylogeny was overall robust, the high degree of conservation of 16S rRNA sequences did not allow to assign strains BDU5 and TSV 85 any clade of Burkholderia

spp. To increase the resolution of this analysis, we repeated it with seven housekeeping genes employed in the Multilocus Sequence Typing (MLST) of the Bcc group (Spilker et al., 2009) (Figure 3.2).

Figure 3.2 Multi-locus sequence typing phylogeny of Burkholderia spp.

Neighbor-joining phylogeny inferred from concatenated protein sequences (4,458 characters) of housekeeping enzymes AtpD, GltB,

GyrB, RecA, LepA, PhaC, and TrpB. Sequences from P. phenazinium LMG 2247T were used as an outgroup. Strains carrying phenazine biosynthesis genes are highlighted in bold font. Indels were ignored in the analysis, and evolutionary distances were estimated using the Jukes-Cantor genetic distance model. The reproducibility of clades was assessed by bootstrap resampling with

1,000 pseudoreplicates, and bootstrap values greater than 60% are indicated by gray circles at the nodes (circle sizes are proportional to bootstrap values). The branch lengths are proportional to the amount of evolutionary change. The scale bars indicate substitution per site.

The dataset included concatenated amino sequences of the products of atpD (ATP synthase beta chain), gltB (glutamate synthase large subunit), gyrB (DNA gyrase subunit

B), recA (recombinase A), lepA (GTP binding protein), phaC (acetoacetyl-CoA reductase), and trpB (tryptophan synthase subunit B). The resultant well-resolved phylogeny agreed with the16S rRNA gene analysis, and also unambiguously identified strains BDU5, BDU6, and TSV 85 as members of the P. pseudomallei clade (Figure 3.2).

The MLST-based phylogeny also confirmed that most Phz+ strains of the Bcc clade were closely related to the primary species of the genus, B. cepacia, and revealed that other phenazine-producing strains clustered with type strains of B. lata, B. pyrrocinia, and B. ubonensis.

Figure 3.3 Contrasting phylogenies using phz proteins and housekeeping enzymes

In some species of Burkholderia phenazine genes may have been acquired via horizontal gene transfer. Contrasting neighbor-joining phylogenies inferred from concatenated sequences of PhzA/B, PhzE, and PhzG proteins (A) and housekeeping enzymes AtpD, GltB,

GyrB, RecA, LepA, PhaC, and TrpB (B). Indels were ignored in the analysis, and the concatenated Phz and housekeeping data sets contained 1,024 and 4,458 characters, respectively. Sequences from P. phenazinium LMG 2247T were used as an outgroup.

Evolutionary distances were estimated using the Jukes-Cantor genetic distance model. The reproducibility of clades was assessed by 24

bootstrap resampling with 1,000 pseudoreplicates, and bootstrap values greater than 60% are indicated by grey circles at the nodes

(circle sizes are proportional to bootstrap values). The branch lengths are proportional to the amount of evolutionary change, and the scale bars indicate substitution per site.

Finally, we attempted to gain insight in the evolutionary trajectory of phenazine genes by contrasting the phylogeny of concatenated sequences of PhzA/B, PhzE, and PhzG proteins to that of the core Burkholderia genome estimated using seven conserved housekeeping genes. This analysis produced phylogenetic trees with mostly congruent topologies (Figure 3.3). One notable exception was discovered in strains B. ubonensis

MSMB 2058 and B. ubonensis MSMB 2035, which are members of the Bcc group, but harbor phenazine biosynthesis genes that are close to those from B. singularis TSV 85, which belongs to the P. pseudomallei clade. Also, the core phenazine genes of BGR1 and

LMG 2196 clustered tightly within the Bcc clade, although both strains are members of the distinct B. glumae/B. gladioli clade.

Figure 3.4 Gene organization of various Burkholderia phz loci

Comparison of the phenazine gene cluster of B. lata ATCC 17760 to its counterparts from other Burkholderia and P. phenazinium, and to the esmeraldin biosynthesis (esm) locus of Streptomyces antibioticus Tu2706. Homologous genes are indicated by arrows of the same color and connected with shading, whereas unique species-specific genes are shown by open arrows. The arrows with bold outlines indicate known or putative phenazine transport genes. The sizes of genes and intergenic regions are not to scale. Locus tags are shown using a code (e.g., the locus tag for the pcm1 homolog of Burkholderia sp. BDU5 is WS69_11045).

We also compared the overall organization and diversity of the phenazine pathways in

Burkholderia spp. A comparison of genome sequences revealed the presence of three distinct types of phenazine cluster (Figure 3.4). The first type is represented by a nine- gene operon found in B. lata ATCC 17760, most strains of the Bcc group, and B. glumae.

The second type was discovered in strains BDU5 and BDU6, where the DNA segment encoding the phzC and pcm3 genes is flipped. The third type of the phenazine locus was identified in B. ubonensis strains MSMB 2058 and MSMB 2035 and in B. singularis

TSV 85. These strains shared an 18-gene phenazine cluster that was very different from its counterparts in other Burkholderia and closely resembled genes involved in the synthesis of phenazine esmeraldin from Streptomyces antibioticus (Rui et al., 2012).

Interestingly, in B. ubonensis MSMB 2035, the phenazine locus is encoded by a conjugative plasmid pMSMB 2013 (GenBank acc. no. NZ_CP013415) that forms part of this strain’s genome. The production of phenazine appears to be a lot less common in the closely related genus Paraburkholderia, and my analysis identified only one Phz+ member of this group, P. phenazinium. The chromosome-encoded phz cluster of this strains is distinct from its counterparts present in genomes of Burkholderia spp. Finally, we also compared the genome regions flanking the phenazine genes in different strains of

Burkholderia. This analysis revealed that in members of the Bcc group, the phz clusters are highly conserved but embedded in different spots of the genome, often adjacent to mobile genetic elements (Figure 3.5).

Figure 3.5 Flanking regions surrounding the phz loci

Synteny of genomic regions carrying genes involved in the synthesis of phenazines in selected strains of Burkholderia. DNA sequences were aligned in Geneious 10.2.3 using MAFFT 7.309 with default parameters. Predicted genes and their orientation are shown by horizontal arrows with phz genes highlighted in red and homologous flanking genes indicated by different colors. All genes and intergenic regions are drawn to scale, as indicated by the scale bar in the upper right corner.

3.2 Genetic analysis of phenazine biosynthesis in B. lata ATCC 17760

The project was initiated by analyzing the spectrum of metabolites produced in strains carrying the full complement of phenazine biosynthesis genes of B. lata. The analysis of fractionated organic solvent extracts from the wild-type strain B. lata ATCC

17760, its complemented mutant B. lata phzA/pBBR1MCS-all, and P. fluorescens 2-79Z carrying a complete set of phz genes on the plasmid pUCP26-all identified 4,9- dihydroxyphenazine-1,6-dicarboxylic acid dimethylester as the final product of the pathway and revealed the presence of multiple phenazine intermediates (Figure 3.6). The synthesis of phenazines in B. lata was strongly affected by the growth conditions, and best yields were observed in cultures grown in King’s medium B (King et al., 1954). The levels of different phenazine derivatives were very dynamic (especially in the overproducer B. lata phzA/pBBR1-MCS-all) and may reflect an inherent feature of the regulation of phz genes in Burkholderia spp. All identified phenazine derivatives had 27

close retention times (7-12 min) on the Acquity UPLC BEH C18 column (502.1mm, diameter of BEH particles 1.7 μm) (Waters) and were therefore difficult to separate. In total, six phenazine compounds (①, ②, ③, ⑤, ⑦ and ⑧, where ① = ⑤) were detected, four of which were purified and characterized in detail by NMR. Two other phenazine compounds (③ and ⑧) could not be isolated in sufficient quantities and were identified based on their HPLC-UV-MS profiles. In addition, one more metabolite (⑥) was provisionally identified as a phenazine derivative, but more data is needed to confirm its structure. The analysis also identified three non-phenazine compounds, ⑨, ⑩, and

⑪, that were produced by the wild-type B. lata but absent from its phenazine-deficient mutants phzA and pcm1. The analysis of isogenic mutants of B. lata revealed that the interruption of phzA, pcm1 and pcm3 genes abolishes the production of all phenazines, which coincides with the loss of purple pigmentation (Figure 3.7). Probing derivatives of

P. synxantha 2-79Z carrying different plasmid deletion variants of the phz locus by

HPLC-MS correlated the presence pcm1 and pcm2 genes with modifications of phenazine-1,6-dicarboxylic acid (PDC), which is the product of the core part of the pathway in B. lata. Specifically, pcm2 was associated with the methylation of carboxyl moieties in positions 1 and 6, while pcm1 was required for the hydroxylation of phenazine ring on positions 4 and 9. Subsequent protein structure predictions with Phyre2 supported these observations and identified the products of Pcm1 and Pcm2 as a flavin monooxygenase and SAM-dependent methyltransferase, respectively. The product of the third putative modifying gene, pcm3, was identified as a NAD(P)H-dependent oxidoreductase, but its exact role in the conversion of PDC to 4,9-dihydroxyphenazine-

1,6-dicarboxylic acid dimethylester remains unclear. 28

Figure 3.6 Phenazine intermediates identified from culture of B. lata ATCC 17760

(A) Phenazine intermediates and other secondary metabolites identified in extracts of B. lata ATCC 17760 (A), and (B) comparison of phenazines produced by B. lata and P. fluorescens 2-79Z carrying the pUCP26-all plasmid. All phenazine derivatives are shown in blue, while the non-phenazine compounds are shown in green. Intermediates ③ and ⑧ could not be purified and were identified based on their HPLC-UV-MS profiles.

Figure 3.7 Genetic organization of phz gene cluster in B. lata ATCC 17760

(A) Genetic organization of the phz gene cluster from B. lata ATCC 17760, isogenic mutants, and pUCP26-based deletion plasmids used in this study. Predicted genes are shown by colored arrows. DNA fragments contained in plasmids used in this study are indicated by thick lines. , insertions of the Tpr cassette in the genome of B. lata; , positions of the lac promoter from pUCP26. The right panel depicts structures of phenazines detected in P. synxantha 2-79Z carrying different deletion plasmids by HPLC-coupled

ESI-MS. (B) The proposed role of enzymes encoded by pcm1 and pcm2 in the biosynthesis of 4,9-dihydrophenazine-1,6-dicarboxylic acid dimethylester. (C) The the appearance of wild-type B. lata ATCC17760 and its phzA mutant in King’s medium B.

3.3 Biological function of phenazine compounds in B. lata ATCC 17760

The analysis of the possible role of phenazines in the virulence of Burkholderia involved two sets of experiments. First, different phenazine-producing and nonproducing strains were compared for the capacity to kill fruit flies in the pricking assay of

Castonguay-Vanier et al. (2010). Results of these assays revealed that Burkholderia readily infect and kill D. melanogaster (Figure 3.8). With the exception of less virulent

Figure 3.8 Survival curves for D. melanogaster infected with Burkholderia spp.

Pricking assays were performed with inocula adjusted to 108 CFU mL-1. Flies in the uninoculated control were pricked with a needle dipped in 10 mM MgSO4. *Significantly different (according to the Kaplan-Meier log-rank test at P ≤ 0.05) from the positive control

ATCC 25416. The experiment was conducted twice with similar results.

B. ambifaria, all tested wild type strains were lethal to flies and killed all inoculated animals within 40 hrs. In contrast, the survival of the uninoculated control (10 mM

MgSO4) was over 86 % (Figure 3.8). The virulence did not appear to correlate with the presence of phz genes, as phenazine-nonproducing B. cepacia ATCC 25416 killed flies on a par with its phenazine-producing (ATCC 17760, PC17, PC39, 2424) and overproducing (phzA/pBBR1MCS-all) counterparts. In the second series of experiments, an attempt was made to slow the rate of killing by lowering the concentration of bacteria used to inoculate fruit flies. Since most Burkholderia were highly virulent toward D. melanogaster, the concentration of inoculum was optimized in a dose-response experiment, where flies were inoculated with suspensions of B. cepacia ATCC 25416 and

B. lata ATCC 17760 adjusted to 107, 106, and 105 CFU mL-1. Results of these assays revealed that lower infection dose resulted in better survival of fruit flies, which at 105

CFU mL-1 approached 80 % (Table 3.1).

Table 3.1. Dose-response assays with B. cepacia ATCC 25416 and B. lata ATCC 17760.

Treatment Inoculum (CFU mL-1) % survival P value B. cepacia ATCC 25416 107 25.0 - 106 55.0 0.1370 105 80.0 0.0017* B. lata ATCC 17760 107 20.0 - 106 60.0 0.0067 105 80.0 0.0012*

*Significantly different from the inoculum level of 107 CFU mL-1 (P ≤ 0.05). The experiment was conducted twice with similar results.

The third round of experiments compared the virulence of the wild-type parental strain B. lata ATCC 17760, its isogenic mutants phzA, pcm1, and pcm3, and the phenazine-overproducing variant harboring the plasmid pBBR1MCS-all. The strains were applied at two inoculum levels: high (107 CFU mL-1) and low (105 CFU mL-1)

(Table 3.2). The side-by-side comparison of ATCC 17760 and its phenazine-deficient and -overproducing derivatives showed no statistically significant difference between treatments thus suggesting against the involvement of phenazines in the virulence towards fruit flies.

Table 3.2. Fruit fly pathogenicity assays with B. lata ATCC 17760 and its isogenic phenazine mutant derivatives.

Inoculated at 107 CFU Inoculated at 105 CFU Treatment a mL-1 mL-1 % survival a P value b % survival a P value b ATCC17760 (wild type - 76.7 - 3.3 control) phzA 26.7 0.8933 86.7 0.3193 pcm1 20.0 0.8435 86.7 0.3611 pcm3 20.0 0.2495 73.3 0.1296 phzA (pBBR1MCS-all) 20.0 0.4989 86.7 0.3144 a Percent survival after seven days. b Comparison to the wild-type control strain using the Kaplan-Meier log-rank test. The experiments were repeated with similar results.

The four phenazine-producing and two nonproducing strains of Burkhoderia were also compared for their capacity to invade plant tissues and cause maceration of onion bulbs. The Phz- B. cepacia ATCC 25416 is a postharvest onion pathogen and was included as a positive control. Results of these assays separated the tested strains into two groups that exhibited, respectively, the high (ATCC 25416, PC39, PC17, 2424) and low

(ATCC 17760 and AMMD) capacity to infect and damage onion bulbs (Table 3.3, Figure

3.9). However, as in the case fruit fly assays, no correlation was observed between the presence of phenazine biosynthesis genes and the degree of onion tissue maceration.

Table 3.3. Onion tissue maceration assays with different Burkholderia spp.

Onion maceration score Strain Experiment 1 Experiment 2

B. cepacia ATCC 25416 (Phz-) 2.3  1.1 A 2.4  0.8 AB

B. ambifaria AMMD (Phz-) 0.4  0.5 B 0.8  0.5 C

B. lata ATCC 17760 (Phz+) 0.4  0.5 B 0.8  0.5 C

Burkholderia sp. PC17 (Phz+) 2.2  0.8 A 2.6  0.5 A

Burkholderia sp. PC39 (Phz+) 1.6  0.9 A 1.8  0.8 AB

Burkholderia sp. 2424 (Phz+) 1.6  1.1 A 1.6  0.9 BC

Onion tissue damage was scored on a scale of 0-3 (0, no visible maceration; 1, < 33% maceration; 2, 34-65% maceration; 3, 66-100% maceration). ATCC 25416 is an onion pathogen and was used as a positive control, and the negative control was inoculated with TSB.

The experiment was conducted twice with five replicates per treatment. Mean comparisons among treatments were performed with

Fisher’s protected LSD test (P ≤ 0.05).

Figure 3.9 Onion tissue maceration by B. cepacia ATCC 25416

The appearance of onion bulb tissue after inoculation with B. cepacia ATCC 25416 (positive control) (A) or Tryptic Soy Broth

(negative control) (B).

In fluorescent Pseudomonas, the formation of biofilms and their morphology is affected by the presence and nature of phenazine compounds. Many species of the B. cepacia complex are known for their capacity to form extensive biofilms, which prompted us to test the possible link between the synthesis of phenazines and formation of biofilms in B. lata. The biofilm assays compared the wild-type strain ATCC 17760, its phenazine-deficient phzA mutant, and the complemented mutant phzA(pBBR1MCS-all), which overproduces phenazines. The bacteria were inoculated in a flow cell connected to a BioFlux 200 microfluidistic system and cultured in a medium conducive for the production of phenazines, King’s medium B. The development of biofilms was monitored over 48 h by acquiring bright field micrographs and subsequently analyzing them in the BioFlux Montage software. Results of these experiments revealed distinct differences in the dynamics of the establishment and development of biofilms in the three tested strains. In particular, the wild-type and overproducer strains attached faster and formed thicker biofilms than the phzA mutant (Figure 3.10). Interestingly, the phenazine- overproducing variant formed the densest biofilms that also did not slough off in the medium flow, whereas mature biofilms of the wild-type strain readily dispersed. A similar trend was observed in static biofilm assays performed with the wild type B. lata strain and its phenazine-deficient and complemented derivatives (Figure 3.11). The crystal violet staining of biofilms formed in PVC plates revealed that ATCC 17760 and phzA(pBBR1MCS-all) bound significantly more dye (P < 0.05 by Kruskal Wallis rank sum and Dunn tests) than the phzA, pcm1, and pcm3 mutants. The most robust biofilm was observed in the complemented phzA mutant, thus mirroring the results obtained in the BioFlux flow cell system.

Figure 3.10 Graph of % coverage of biofilm in BioFlux microfluidistic system

Continuous monitoring of biofilm formation by B. lata ATCC 17760, its phenazine-deficient phzA mutant, and the phenazine- overproducing derivative phzA(pBBR1MCS-all). The three stains were inoculated into the BioFlux microfluidic system and allowed to form a biofilm for 48 h. The bright-field images were collected at 20 min intervals with a LS620 digital microscope. The top panel indicates the surface area covered by the growing biofilm, whereas the bottom panel shows representative images of biofilm development over the course of the experiment.

Figure 3.11 Static biofilm formation

Static biofilms formed by B. lata ATCC 17760 and its phenazine-deficient and complemented derivatives. All strains were inoculated at approximately 106 CFU mL-1 and grown in 96-well PVC plates for 48 h, at which point the biofilms were stained with crystal violet. Bars with different letters indicate significant differences as determined by the Kruskal-Wallis rank test followed by Dunn’s multiple comparisons test (P < 0.05).

CHAPTER IV – DISCUSSION

To the best of our knowledge, this is the first study that attempted to comprehensively address the genetics, evolution, and biological role of phenazine compounds in Burkholderia. Although the production of phenazines in this economically-important group of microorganisms was first reported almost 70 years ago

(Turner & Messenger, 1986), the access to recently generated genome sequences allowed us for the first time to assess the distribution, organization of biosynthesis gene clusters, and habitats that support in this versatile and economically important group of phenazine- producing bacteria. Our results revealed that phz genes are present in genomes of many

Burkholderia that have a worldwide origin and belong to different species of the genus.

Most Phz+ strains were in the Bcc group, but the capacity to synthesize phenazines was also found in some isolates of the B. pseudomallei clade and the plant pathogen B. glumae. Within the Bcc group, most phenazine producers clustered closely with B. cepacia thus agreeing with the findings of Mavrodi et al. (2010), who identified several strains of Phz+ Burkholderia from Japan as B. cepacia based on amplicon sequences of phzF, 16S rRNA, and recA. We also confirmed the presence of phz genes in B. lata

(ATCC 17760), B. pyrrocinia (strain 5.5B), and identified the first phenazine-producing strains of B. ubonensis (MSMSB 2035 and MSMB 2058). Outside of the Bcc complex, we discovered the first gene clusters in members of the P. pseudomallei group, including

B. singularis TSV85 and two strains, BDU5 and BDU6, that did not align closely with type strains and may represent new species of this clade. Finally, we confirmed the presence of phz genes in genomes of plant-pathogenic B. glumae.

Most of the phenazine producers studied in this work were isolated from the environment, namely soil, but some originated from diseased plants. Although listed as saprophytes, many strains of Phz+ Burkholderia were closely related to species that are known to enclose numerous genetically close environmental and clinical isolates.

Furthermore, several Phz+ strains belonged to P. pseudomallei and B. glumae groups that encompass serious pathogens of humans and animals, and crop plants (Eberl &

Vandamme, 2016). Interestingly, phenazine production seems to be a common trait in B. glumae since a study of strains native to the southern United States identified several virulent isolates that produced different pigments, one of which was later identified as the phenazine phencomycin (Karki et al., 2012; Han et al., 2014). Therefore, the question of the association of phenazine production with the saprophytic and parasitic lifestyles of

Burkholderia spp. clearly deserves further investigation.

While conducting this study, we were surprised to discover that the majority of

Phz+ Burkholderia with sequenced genomes were isolated in Australia. It is plausible that this prevalence of Australian strains simply reflects a non-random sampling since

Phz+ Burkholderia were previously isolated in the US (Karki et al., 2012; Cartwright et al., 1995), Japan (Seo & Tsuchiya, 2004), Europe (Smirnov & Kiprianova, 1990), and other parts of the world (Stanier, Palleroni, & Doudoroff, 1966). Alternatively, it is possible that phenazine-producing strains of Burkholderia are more common in specific locations, similar to some groups of Phz+ pseudomonads that are enriched in arid soils of the US Pacific Northwest (Mavrodi et al., 2012a; Parejko et al., 2012) and Fusarium wilt-suppressive soils of Chateaurenard, France (Mazurier et al., 2009). Mavrodi et al.

(2012) further demonstrated the involvement of these bacteria in the suppression of

Rhizoctonia root rot disease and the presence of nanomolar levels of phenazine antibiotics in the rhizosphere of field-grown wheat. Further studies also revealed the strong impact of irrigation and precipitation on indigenous populations of phenazine- producing Pseudomonas in dryland wheat fields of central Washington and Oregon

(Mavrodi et al., 2012b; Mavrodi et al., 2018). It is tempting to speculate that combination of environmental and edaphic factors may result in the enrichment of Phz+ Burkholderia in certain parts of the world, although further research is needed to confirm or reject this hypothesis.

Our study revealed at least three distinct variants of phenazine clusters in phenazine-producing Burkholderia. Most Phz+ species of the B. cepacia and B. glumae clades shared an operon comprised of six core and three modifying biosynthesis genes. A variation of this scheme was observed in strains BDU5 and BDU6, which had a gene cluster with an inverted DNA segment containing the phzC and pcm3 genes. Very different phenazine pathways were identified B. singularis TSV 85 and B. ubonensis

MSMSB 2035 and MSMB 2058. The phz clusters in these species had 18 genes, some of which encoded modifying enzymes similar to those involved in the synthesis of esmeraldin in S. antibioticus (Rui et al., 2012). Another distinct phenazine cluster was discovered in P. phenazinium, which differed from its Burkholderia counterparts in the number of core and modifying genes. Overall, these pathways differed in the number and types of predicted phenazine-modifying and efflux genes. All Phz+ Burkholderia also contained a full complement of core biosynthesis genes, although their arrangement and even structure varied between species of phenazine producers. In particular, genomes of

TSV 85, MSMSB 2035, and MSMB 2058 encoded a Pseudomonas-like version of PhzF

that lacked the 120-amino acid N-terminal domain, which is present in homologous proteins of other Burkholderia spp. These strains also carried an unusually long version of the isochorismatase PhzD, which represents a fusion of the S-adenosylmethionine- dependent methyltransferase and isochorismate hydrolase domains. Finally, the phz clusters of TSV 85, MSMSB 2035, and MSMB 2058 lacked the phzC gene, but at the same time carried a gene encoding a canonical 3-deoxy-7-phosphoheptulonate synthase.

We hypothesize that this enzyme compensates for the lack of PhzC and aids the synthesis of phenazines by provides metabolic precursors for the shikimic acid pathway. Our results also revealed the conservation of phz clusters in most species of the Bcc group, as well the evidence for horizontal gene transfer in B. glumae and strains TSV 85, MSMSB

2035, and MSMB 2058, which harbor phenazine genes on conjugative plasmids.

Although structurally conserved, the phenazine operons of Bcc strains are located in different genomic regions, which is likely a result of genomic relocation. Collectively these findings suggest that phenazine biosynthetic pathway of Burkholderia resembles its counterpart from Pseudomonas (Fitzpatrick, 2009; Mavrodi et al., 2010; Biessy et al.,

2018) in that it has a complex evolutionary history, which likely involved horizontal gene transfers among several distantly related groups of producing organisms.

Although structures of some phenazines produced by Burkholderia spp. were elucidated several decades ago, the genetic basis for this process remained unexplored.

Our experiments with B. lata ATCC 17760 represent the first formal confirmation of the involvement of the phz cluster in the production of 4,9-dihydroxyphenazine-1,6- dicarboxylic acid dimethylester. Furthermore, our assays with plasmid deletion derivatives provide the first experimental evidence for the role that the three pcm genes

play in the modification of the phenazine tricycle during the synthesis of 4,9- dihydroxyphenazine-1,6-dicarboxylic acid dimethylester. Overall, our results agree with the findings of Korth et al. (1978) and indicate the production of phenazine in Bcc strains is a highly dynamic process. The presence of multiple intermediates probably reflects the symmetrical nature of the final phenazine product, which is assembled via sequential hydroxylation and methylation of the PDC precursor. The dynamic nature and yield of different phenazine derivatives suggest the complex effect of environment and specific and global regulation, details of which remain entirely unknown.

Earlier studies reported the broad antibiotic activity of phenazines produced by several species of the genus Burkholderia (Smirnov & Kiprianova, 1990; Cartwright et al., 1995; Han et al., 2014). However, recent studies in P. aeruginosa prompted us to probe the broader biological role of phenazines in this diverse group of bacteria.

Although we failed to correlate the production of phenazines with the capacity of Phz+

Burkholderia to kill fruit flies and rot onions, other experiments revealed a link between the presence and amount of phenazines and the dynamics of biofilm growth flow in both the flow cell and static experimental systems. These preliminary findings suggest that

Burkholderia, like fluorescent pseudomonads, may benefit from the unique redox-cycling properties of phenazines and their capacity to act as extracellular electron shuttles in biofilms. These findings also suggest that the contribution of phenazines to the pathogenicity of Phz+ members of the Bcc group should be revisited using chronic models of infection that often involve the biofilm mode of growth.

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