Exploring the Genetic Mechanisms and Community Dynamics of Bacterial Social Behaviors Making Rhodobacterales Sp. Y4I a Competitive, Dominant Member of Marine Microbial Synthetic Communities.














A Thesis Presented for the
Doctor of Philosophy
Degree
The University of Tennessee, Knoxville













April C. Armes
December 2021
Copyright © 2021 by April Armes.
All rights reserved.















DEDICATION

To my parents, Ron and Tina Mitchell. They would have been so proud. 





ACKNOWLEDGEMENTS


I have a lot of people to thank but most importantly, I want to acknowledge my cat, Levi. Without him, none of this work would be done. He basically carried me through grad school. 





ABSTRACT


This dissertation addresses the underlying genetics regulating competitive physiologies controlled by QS in a dominant member of marine synthetic communities. Furthermore, it addresses whether QS affects marine microbial biofilm community structure and dynamics. Finally, I provide an in depth review of the breadth of QS homologs within the Roseobacter lineage and identify a clade-specific DNA regulatory sequence (‘roseobox’). Using this roseobox, I identify putative gene pathways regulated by QS in environmental sequence data. 



TABLE OF CONTENTS

Chapter 1 INTRODUCTION	2
I.	Background	2
II. Bacterial Biofilms	2
II.	The Roseobacter lineage.	3
III.	Research Objectives	5
References	6
Chapter 2 Cyclic di-GMP is integrated into a hierarchal quorum sensing network regulating antimicrobial production and biofilm formation in Roseobacter clade member	8
I.	Abstract	9
II.	Introduction	9
III.	Materials and Methods	10
IV.	Results	12
V.	Discussion	14
VI.	References	16
Appendix: Tables	19
Appendix: Figures	20
Appendix: Supplemental Materials	25
Chapter 3 Quorum Sensing And Carbon Source Influence Biofilm Structure In Marine Roseobacter Synthetic Communities	31
I.	Abstract	32
II.	Introduction	33
III.	Materials and Methods	33
IV.	Results	35
V.	Discussion	35
References	36
Appendix: Tables	37
Appendix: Figures	39
Appendix: Supplemental Materials	46
Chapter 4 : Quorum Sensing in Roseobacters: Clade specific Roseobox identifies genes regulated by quorum sensing.	47
I.	Abstract	48
II.	Introduction	49
III.	Materials and Methods	50
IV.	Results	50
V.	Discussion	50
VI.	References	51
Appendix: Figures	51
Chapter 5 CONCLUSION	52
VITA	53



LIST OF TABLES

Table 2.1 Y4I variant phenotypes	26
Table 3.1. Table of community members, their luxRI homologous genetic loci, and putative AHLs produced by community members.	53
Table 3.2. Factorial growth inhibition of synthetic community members.	55
Table 4.1 Positive matches to roseo box motif 1	76
Table 4.2 Positive matches to roseo box motif 2	91

LIST OF SUMMPLEMETAL TABLES

Table S 2.1. Table of primers.	25
Table S 2.2. Position and nature of insertional mutations and location of qPCR primers.	26










LIST OF FIGURES

Figure 2.1 Gene expression of luxRI homologs and igiD synthase from agar grown cultures..	24
Figure 2.2 Indigoidine production in response to growth-phase dependent AHL exogenous addition. Indigoidine quantification in Y4I variants	25
Figure 2.3 Surface Colonization and Biofilm production over time in Y4I mutants..	26
Figure 2.4 Cyclic-di-GMP concentrations in broth and agar grown cultures..	27
Figure 2.5 Working model of the regulatory network governing key physiologies in strain Y4I.	28
Figure 3.1. Surface colonization of Roseobacter strains to glass beads in a complex medium over 48 hrs.	43
Figure 3.2. Relative abundance of community members over time of biofilms grown in complex medium (A) and defined medium (B)..	44
Figure 3.3. PCoA plot using Bray Curtis Dissimilarity Index to determine beta diversity between each carbon source, mixed community, and time point..	45
Figure 3.4 Biofilm production of monoculture community members in complex medium and five-member synthetic communities over time (A-C)..	47
Figure 3.5 Biofilm production of monoculture community members and five-member synthetic communities over time (A-D) in defined medium (2 mM coumarate)..	49

LIST OF SUPPLEMENTAL FIGURES

Figure S 2.1 Overview of methodology for AHL add back..	27
Figure S 2.2. Growth dynamics in liquid culture (A) and biofilm formation (B) in Y4I and mutants.	28
Figure S 2.3. Growth dynamics in liquid culture (A) and biofilm formation (B) in Y4I and mutants.	28
Figure S 2.4. Half CtrA binding site within pgaRI (green) operon.	28






1
A version of this introduction was originally written by Alison Buchan, April Mitchell (Armes), W. Nathan Cude, and Shawn Campagna and is published in Stress and Environmental Regulation of Gene Expression and Adaptation in Bacteria.
	
Alison Buchan, April Mitchell, W. Nathan Cude, and Shawn Campagna. “Acyl-homoserine lactone-based quorum sensing in members of the marine bacterial Roseobacter clade: complex cell-to-cell communication controls multiple physiologies” Stress and Environmental Regulation of Gene Expression and Adaptation in Bacteria (2016): 225-233

My contribution to this publication was to provide an updated review of the role of quorum sensing in Roseobacter biology. In this review, I provided discussion of the integration of the master regulatory, CtrA, into the Roseobacter QS regulatory network; and expanded upon the current knowledge of QS in Roseobacters.  


INTRODUCTION

I. Background
Bacteria have evolved a variety of genetic mechanisms for behaviors in response to environmental signals. One of these mechanisms, frequently referred to as quorum sensing (QS), is dependent upon the concentration of bacterially produced, diffusible chemical cues. Bacteria capable of producing these signal molecules release them into the local environment where they can be sensed by bacteria of the same or different species resulting in regulation of gene expression in a manner that depends upon signal concentration and potentially other environmental cues. In this way, bacterial communities use these signaling systems to communicate, cooperate and/or compete with one another as a form of social interaction. It is hypothesized that QS allows these bacterial consortia to behave as pseudo-multicellular organisms. Phylogenetically diverse bacteria use QS systems to coordinate gene expression (1, 2). QS networks have been implicated in bacterial behaviors including bioluminescence, host colonization, biofilm formation, coordinated motility (i.e., swarming) and virulence (1). 
Several classes of small molecule signals are known to mediate intraspecies signaling in bacteria. These signals often upregulate their own production and have been termed autoinducers (1, 3, 4). The major class of intraspecies signal in Proteobacteria are N-acylhomoserine lactones (AHLs) (2). Canonical AHL-QS systems produce and respond to AHLs via two proteins, LuxI and LuxR, respectively. The genes encoding these proteins are most often located adjacent to one another on the bacterial chromosome in alphaproteobacteria (1). LuxI family proteins synthesize AHLs via cyclization of S-adenosyl methionine, forming a lactone ring, to which an acylated carbon chain, derived from fatty acid biosynthesis, is conjugated (3). Acyl chain length and saturation, combined with the presence or absence of oxidation at the third carbon, by either a hydroxyl or carbonyl group, allows for species or group specificity (1, 3). LuxR proteins represent a large family of proteins, all of which are response regulators (5). Those LuxR proteins that function in QS systems mediate gene expression required for communal behavior in response to intracellular concentrations of their cognate AHLs (5, 6). Activated LuxR proteins, that is LuxRs bound to their cognate AHLs, typically upregulate luxI transcription by binding to a DNA regulatory element termed ‘lux box’ which enhances the rate of AHL synthesis. Activated LuxR proteins also modulate expression of other genes, including those involved in secondary metabolite production, motility, biofilm formation, and others (1, 2, 5). 
II. Bacterial Biofilms 
The dominant lifestyle of bacteria in environmental ecosystems takes the form of a biofilm. Biofilms consist of a microbial consortium suspended in an extracellular polymeric substance. Biofilm formation begins by the sensing of a surface, biotic or abiotic in nature. This action, usually referred to as mechanosensing, typically involves external cues such as torque of a flagellum as it encounters a surface (7). Involved in this “swim-stick” mechanism is typically a response regulator that is activated by environmental stimuli. In the alpha-proteobacterium, Caulobacter crescentus, this response regulator is known as the master regulator CtrA. In addition to the “swim-stick” switch, the CtrA phosophorelay system also regulates cell cycle, polar cell division, and flagellar biosynthesis (8–10). This system is highly conserved amongst Alphaproteobacteria and its role in lifestyle switch has been demonstrated in several Roseobacters (discussed further below)(11). 
Another regulator of biofilm formation is the global secondary messenger bis-(3′-5′)-cyclic dimeric guanosine monophosphate (c-di-GMP). As with the CtrA phosphorelay system, in response to environmental stimuli intracellular, as levels of c-di-GMP reach a concentration threshold induction of genes, including those promoting biofilm formation, are upregulated (12). C-di-GMP can regulate the production of most known extracellular matrix components on transcriptional, post transcriptional, or posttranslational levels (11). Furthermore, central metabolism and intracellular pools of c-di-GMP have been linked through glutamate-induced cyclase activity in some bacteria (e.g., Pseudomonas aeruginosa (13)). In a group of marine Alphaproteobacteria, known as the Roseobacter Clade, c-di-GMP has been suggested to regulate biofilm formation, likely through the regulation of the CtrA phosphorelay system and integration of cell-to-cell QS (14, 15).
III. The Roseobacter lineage 
Members of the Roseobacter clade fall within the Rhodobacteraceae family of the Alphaproteobacteria class and are well represented across diverse marine habitats, from coastal to open oceans and from sea ice to sea floor. The lineage encompasses a large, phylogenetically diverse group of bacteria.  At present, the clade contains well over 50 described genera and thousands of uncharacterized isolates and clone sequences (16). It has been proposed that the clade can be parsed into five deeply branching subclades based on a phylogenetic analysis of 70 concatenated conserved single-copy genes (17). The first described members of the group were Roseobacter litoralis and Roseobacter denitrificans, both pink-pigmented bacteriochlorophyll-a producers (18).  Subsequent cultivation of clade members, however, revealed that most strains are neither pink nor bacteriochlorophyll-a producers.  With very few exceptions, the Roseobacter clade is exclusively marine or hypersaline (16).  High natural abundances of Roseobacters were first demonstrated in the coastal waters of the southeastern US in the late 1990s using culture-independent approaches (19).  Subsequent studies based on culture collections, 16S rRNA gene clone libraries, and single cell analyses, reveal that Roseobacters are found in almost all marine environments sampled (20). Molecular-based approaches targeting 16S rRNA genes demonstrate that the Roseobacter clade is one of the major marine bacterial groups, typically comprising upwards of 20% of coastal and 15% of mixed layer ocean bacterioplankton communities (20).  Members have been found to be free-living or particle-associated, or in commensal relationships with marine phytoplankton, invertebrates and vertebrates (20).  Furthermore, representatives of this clade stand out as one of the most readily cultivated of the major marine lineages (21). These isolated representatives are serving as the foundation for an improved understanding of marine bacterial ecology and physiology. Many of these studies are facilitated by the wealth of genome data that is available for both cultured and uncultured representatives.
Genome sequences of cultivated Roseobacter strains reveal relatively large genomes (~4.5 Mb) with gene content that suggests versatility may be driving this cosmopolitan group’s ecological success (22).  Evidence from environmental, microbiological, genomic and metagenomic studies point to the involvement of members of the Roseobacter lineage in several key biogeochemical processes, including mineralization of plant-derived compounds and transformations of reduced inorganic and organic sulfur compounds (20, 23). Another trait characteristic of Roseobacter clade strains is the production of secondary metabolites, including AHLs (24). Several recent studies have begun to examine the contribution of QS to the physiology of cultivated representatives (discussed below).
The role of quorum sensing in Roseobacter biology. The first evidence of QS in Roseobacters demonstrated AHL production in representative strains isolated from marine snow using an Agrobacterium tumefaciens bioreporter strain (25). Subsequent studies have provided evidence that QS is involved in the colonization of both living organisms and non-living surfaces in marine environments and have shown the involvement of QS in a variety of physiologies, from secondary metabolite production to cellular morphology (26–29). Furthermore, as primary and aggressive surface colonizers, members of the Roseobacter clade are hypothesized to set the stage for microbial biofilm structure, function, and dynamics via QS-mediated interactions (30). Despite the prevalence of QS in this clade, only a few representative Roseobacters have been the primary focus of experimental studies of QS and none of these representatives are currently being utilized to determine the effect of QS on microbe-microbe interactions within a biofilm. 
AHLs characterized from Roseobacter isolates possess some of the longest acyl side chains recognized with lengths ranging from C4 to C18 and possessing differing degrees of saturation and oxidation (24, 31).  Relatively common among Alphaproteobacteria, long chain AHLs are hydrophobic in nature, often causing them to partition in cell membranes where they may not be as readily available as diffusible signaling molecules (32). QS signal efflux pumps have been identified in other Proteobacteria, but similar mechanisms have yet to be identified in Alphaproteobacteria. Thus, the biological implications of membrane “sequestering” of QS molecules are not yet fully appreciated. In addition, it has been noted that long chain AHLs are more stable than their shorter chain counterparts in alkaline environments, such as the world’s oceans. Thus, it has been recently suggested that the slight alkalinity of seawater may be a driving force in shaping the AHL repertoire of Roseobacters (33).  
Culture-based studies indicate that Roseobacters are the primary symbiont producers of AHLs (34). A model for sponge-associated Roseobacter, Ruegeria sp. KLH11, contains two sets of luxRI homologs, designated ssaRI and ssbRI This strain also possesses an orphan, or solo, luxI, designated sscI, in its genome (28, 35). SsaI, SsbI, and SscI predominantly produce long chain saturated and unsaturated AHLs ranging in size from C12 to C16. SsaI produces 3O-AHL variants whereas SsbI and SscI produce 3OH-AHLs (28). The binding affinity of signaling molecules to LuxR homologs is influenced by modifications at the third carbon, and this may allow KLH11 to finely tune its metabolism to cellular density and AHL diversity (36).  Similarly, the algal symbiont Dinoroseobacter shibae DFL12 is another representative of the Roseobacter clade reported to have two luxR-like cassettes as well as an luxI orphan, designated luxRI1, luxRI2, and luxI3, respectively (37). Deletion of luxI1 has been shown to affect the regulation of the other two QS systems, indicating the regulatory network in D. shibae is organized in a hierarchical fashion (38). QS in D. shibae has also been demonstrated to regulate the cckA-chpT-ctrA regulatory circuit, which mediates cell cycle in this strain and influences cellular morphology (38). D. shibae populations show variation in cell morphology which is hypothesized to contribute to fitness in changing environments (39). 
Integration of QS and cell cycle regulatory mechanisms, such as the cckA-chpT-ctrA circuit and the secondary global messenger c-di-GMP, in Roseobacters is an emerging area of research. While the specific mechanistic details of the interactions have yet to be elucidated, it has been shown in D. shibae that not only is the cckA-chpT-ctrA regulatory circuit impaired in QS mutants but that AHL production is impaired in a ctrA mutant (29). Thus, the regulatory network of these two systems is clearly connected, in at least some Roseobacters. The extent to which this integration of networks is present amongst the larger collection of Roseobacters has been probed bioinformatically. CtrA, which functions as a master regulator in many Alphaproteobacteria has a conserved binding site that can be recognized by genome analysis (29). Wang et al. (29), found CtrA binding sites within the luxRI cassettes of five of the ten Roseobacters genomes analyzed. O. antarcticus 307 possesses CtrA binding sites in both of its luxRI cassettes. R. denitrificans, R. litoralis, R. pomeroyi, and D. shibae were all predicted to have a single CtrA binding site within one luxRI cassette found within these strains. Additionally, QS-mediated physiologies, such as surface attachment and biofilm formation, is often modulated through intracellular signaling of the global secondary messenger c-di-GMP. Concentrations of c-di-GMP are often influenced by environmental cues and cell-to-cell signaling in bacteria (11). In Rugeria mobilis, c-di-GMP has been demonstrated to regulate biofilm formation and the production of the QS-mediated antimicrobial, tropodithietic acid (TDA) (40). Thus, there is strong indication that the integration of these networks is common amongst, but not absolutely conserved, in Roseobacters.
AHL-based quorum sensing systems are prevalent in cultured Roseobacter representatives. The inventory of chemical signals produced by Roseobacters is anticipated to be large and result in complex signaling pathways in lineage members.  Some of these pathways may be involved in interspecies interactions that influence biogeochemical cycles. For instance, uncharacterized Roseobacters has been shown to be epibionts of Trichodesmium. AHL-based interactions between Trichodesmium and select epibionts has been shown to stimulate phosphorus acquisition in this marine cyanobacterium (41, 42). Additionally, it has been suggested but not yet demonstrated that QS plays a role in the switch from mutualistic to antagonistic behavior in P. gallaeciensis against the bloom-forming marine phytoplankton species Emiliana huxleyi (43). Furthermore, as critical mediators of the decomposition of vascular plant material in coastal oceans, which is an essential process within the global carbon cycle, Roseobacters colonize and mediate transformation of plant-derived aromatic compounds in marine ecosystems. However, the role of QS in colonization, composition of biofilm community members, and interactions has yet to be elucidated for marine ecosystems. Social behaviors regulated by QS between Roseobacters is anticipated to be essential in the varied relationships these bacteria have with marine primary producers and undoubtedly influences the marine food web as well as Earth’s climate. Given the abundance, genetic repertoire, and metabolic capabilities among Roseobacters, the establishment of a genetically characterized model for use in synthetic community studies is of upmost importance for our understanding of the role QS plays in influencing biofilm dynamics. Moreover, while the breadth of QS in Roseobacters has been probed bioinformatically, attempts to pair QS regulatory elements with physiologies associated with bacterial social behaviors has not yet been made.  
IV. Research Objectives 
Despite numerous studies detailing various physiological aspects of Roseobacter clade members, QS systems have only been a focus in a limited number of strains. Furthermore, relatively little is understood of the genetic and physiological mechanisms underlying biofilm formation and microbial competition within this clade. Due to its prolific surface colonization, dominance in synthetic microbial communities, and ability readily outcompete strains in pairwise and multi-species communities (44, 45), Rhodobacterales sp. Y4I shows promise as an emerging model organism to better understand the roles QS play in Roseobacter physiology. Elucidating the genetic hierarchy regulating competitive physiologies in a member of the Roseobacter clade, such as Y4I, is an important first step to our understanding of how these dominate marine microorganisms regulate social behaviors. Many studies have explored how social behaviors mediated by quorum sensing influence community cooperation and competition within microbial biofilms and the role of these interactions in the diversity and stability of microbial communities (46). However, these studies have failed to encompass ecologically relevant microbial communities and their interactions, especially those in marine ecosystems. Thus, understanding the role of QS in interspecies interactions within marine associated biofilms has been relatively unexplored. Understanding the regulatory elements influencing biofilm community dynamics aids our ability to utilize Roseobacters as model organisms for marine ecology. Bacterial social behaviors, such as community cooperation and competition, within microbial biofilms are often mediated by QS (CITE). QS utilizes a special class of transcriptional regulators with signal molecule and DNA binding domains (CITE). Despite this prevalence for QS in clade members, few studies have demonstrated direct transcriptional regulation of population level behaviors within members of this clade. Recent genomic evidence suggests up to 87% of sequenced Roseobacter genomes contain at least one QS systems (33). Of those, 56% contain multiple at least one copy of each gene and are considered capable of QS-mediated behaviors (47). Yet the bioinformatic tools available to identify DNA regulatory elements, such as the lux box, within the Roseobacter clade are lacking. The establishment of clade-specific regulatory element (termed ‘roseo box’) will allow us to query genomic data for social behaviors regulated by QS allowing us to design hypothesis-driven experiments with representatives of the environmentally relevant Roseobacter clade.
The work presented in this dissertation broadly seeks to resolve the gaps in knowledge regarding the regulation, interactions, and prevalence of bacterial social behaviors in Roseobacters, using strain Rhodobacterales sp strain Y4I as a representative for competitive social behaviours. Prior work has demonstrated that QS plays a regulatory role in antimicrobial biosynthesis, but many open questions regarding the multi-layered control system remain. Chapter 2 of this dissertation further characterizes the QS regulatory network governing these competitive social behaviors in Y4I, such as antimicrobial production and prolific surface colonization. In Chapter 3, the contribution of QS and indigoidine production to interactions, such as cooperation and competition, within a five-member mixed species Roseobacter biofilm community is explored. Finally, Chapter 4, uses a bioinformatic approach to examine the prevalence of bacterial social behaviors within the Roseobacter clade by identifying DNA binding regions for LuxR family proteins. Collectively, the work presented in this dissertation provides the necessary framework for understanding bacterial social behaviors and their regulation using a Roseobacter model. 


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Cyclic di-GMP is integrated into a hierarchal quorum sensing network regulating antimicrobial production and biofilm formation in Roseobacter clade member



	A version of this chapter was originally written by April C. Armes and Alison Buchan and has been published by Frontiers in Marine Science:
	
April C. Armes and Alison Buchan. “Cyclic-di-GMP is integrated into a hierarchal quorum sensing network regulating antimicrobial production and biofilm formation in Roseobacter Clade member Rhodobacterales sp. Y4I.” Frontiers in Marine Science (2021). 

A.C.A. and A. B. conceived and designed the experiments for quantitative Reverse-Transcriptase Polymerase Chain Reaction (qRT-PCR) and surface attachment and biofilm formation assays. A.C. A. conceived and designed experiments for acyl-homoserine lactone (AHL) add-back assays and bis-(3’-5’)-cyclic dimeric guanosine monophosphate (c-di-GMP) analysis. A.C.A. conducted the experiments, carried out data analysis, generated figures, and wrote the manuscript. The manuscript was edited by A.C.A and A. B.  

I. Abstract	
	Microbial biofilms associated with marine particulate organic matter carry out transformations that influence local and regional biogeochemical cycles. Early microbial colonizers are often hypothesized to “set the stage” for biofilm structure, dynamics, and function via N-acetylated homoserine lactone (AHL)-mediated quorum sensing (QS). Production of AHLs, as well as antimicrobials, contributes to the colonization success of members of the Roseobacter clade. One member of this group of abundant marine bacteria, Rhodobacterales sp.Y4I, possesses two QS systems, phaRI (QS1) and pgaRI (QS2). Here, we characterize mutants in both QS systems to provide genetic evidence that the two systems work in hierarchical fashion to coordinate production of the antimicrobial indigoidine as well as biofilm formation. A mutation in pgaR (QS2) results in decreased expression of genes encoding both QS systems as well as those governing the biosynthesis of indigoidine.  In contrast, mutations in QS1 did not significantly influence gene expression of QS2. Addition of exogenous AHLs to QS1 and QS2 mutants led to partial restoration of indigoidine production (45-60% of WT) for QS1 but not QS2.  Mutational disruptions of QS1 had a more pronounced effect on biofilm development than those in QS2. Finally, we demonstrate that c-di-GMP levels are altered in QS and indigoidine biosynthesis Y4I mutants. Together, these results indicate that pgaRI (QS2) is at the top of a regulatory hierarchy governing indigoidine biosynthesis and that the global regulatory metabolite, c-di-GMP, is likely integrated into the QS circuitry of this strain. These findings provide mechanistic understanding of physiological processes that are important in elucidating factors driving competitiveness of roseobacters in nature.



II. Introduction
Cell-to-cell signaling known as quorum sensing (QS) allows bacterial populations to coordinate gene expression. This coordination of gene expression typically occurs in a cell density dependent manner, through the production and recognition of small diffusible signaling molecules. A prevalent class of these molecules in Gram-negative Proteobacteria are N-acylhomoserine lactones (AHLs) (1). AHL-based QS characteristically involves a two-component system consisting of a transcriptional regulator (denoted by -R) and an AHL synthase (designated as -I). When bound to their cognate transcriptional regulator, AHLs can elicit global changes in gene expression (2). In model biofilm forming representatives, QS has been shown to genetically regulate surface attachment and biofilm formation (3, 4). Surface attachment and biofilm formation is also often modulated through intracellular signaling of a global secondary messenger of bis-(3’-5’)-cyclic dimeric guanosine monophosphate (c-di-GMP). Concentrations of c-di-GMP are influenced by environmental cues as well as cell-to-cell signaling in bacteria (5). Indeed, c-di-GMP signaling is integrated in QS regulatory systems in model biofilm bacteria, such as Pseudomonas aeruginosa (6). Integration of this intracellular signaling molecule into QS architectures more tightly links cellular physiology with adaptive behaviors. 
	Members of the Roseobacter clade are competitive surface colonizers in diverse marine niches. This lineage of heterotrophic Alphaproteobacteria represent one of the most phylogenetically coherent, yet physiologically diverse, groups of marine bacteria. Roseobacters are among the most abundant, metabolically active, and aggressive surface colonizers in marine ecosystems (7). As such, members of this clade inhabit a wide range of niches and readily colonize surfaces both abiotic and biotic in nature, e.g. sinking particulate organic matter (8), Trichodesmium colonies (9), and polymer test surfaces (10). Recent genomic evidence suggests up to 87% of sequenced Roseobacter genomes contain at least one QS systems. Of those, half contain multiple QS systems (11). Despite the prevalence of QS systems identified in Roseobacter genomes, these systems have been studied in a limited number of strains. 
 Rhodobacterales sp. Y4I shows promise as an emerging model organism to better understand the roles QS play in Roseobacter physiology. Isolated from a coastal marsh ecosystem in the southeastern United States, Y4I has been shown to be a competitive surface colonizer, prolific biofilm former, and a dominant member in mixed species mesocosm experiments (12, 13). Specifically, Y4I has been shown to have a competitive advantage against other marine bacteria, including members of its own clade, when grown on a surface. This fitness advantage has been demonstrated to be linked to the production of the blue-pigmented, redox reactive antimicrobial indigoidine (12). Indigoidine is regulated by two QS systems, phaRI (QS1) and pgaRI (QS2), in this strain. Previous studies demonstrated a disruption of either QS system influences both indigoidine and AHL production. However, the contributions of each QS system to production of indigoidine were ambiguous due to the expression of a leaky phenotype in a QS1 mutant (14). In addition, the regulatory network governing indigoidine biosynthesis is complex, particularly given that surface attachment is a prerequisite for its production, suggesting that cellular mechanisms beyond QS alone may play a role in coordinated cellular responses to environmental conditions. Here, we assess the hierarchical organization of these two QS systems to coordinate production of the antimicrobial indigoidine and biofilm formation. We also present evidence that the secondary global messenger, c-di-GMP, is integrated into the complex regulatory network governing surface attachment and indigoidine production. 

III. Materials and Methods
Strains, growth conditions, and maintenance
Rhodobacterales sp. Y4I was previously isolated from an estuary off the coast of Georgia in the southeastern United States (15, 16). Insertional mutants in either the indigoidine synthase gene, igiD::Tn5-KmR (formerly Y401BD4), or QS transcriptional regulators, phaR::Tn5-KmR (formerly Y412AE6) and pgaR::Tn5-KmR (formerly Y411CE4) were previously generated using a mini Tn5 transposon system (12). Aliivibrio fischeri ES114 (ATCC 700601) was kindly provided by Eric Stabb (University of Illinois).  Unless otherwise noted, all Y4I and A. fischeri strains were routinely grown in YTSS (per liter: 15g Sea Salts (Sigma) or Instant Ocean (Thermo Fisher Scientific), 4g tryptone, 2.5g yeast extract) and incubated at 30°C and 25°C, respectively. Escherichia coli BW20767, which is pir+, was maintained on Luria-Bertani (LB) and routinely grown at 37°C on LB unless otherwise noted (17). 
Construction of phaI mutant using insertional mutagenesis
Insertional mutagenesis of the phaI (QS1) gene of strain Y4I was achieved using the pKNOCK-KmR plasmid (18). Briefly, an internal fragment of phaI (302 bp) was PCR amplified using primers Y4I_3464_for and Y4I_3464_rev  (Table S1)and ligated into the PCR cloning vector TA TOPO pCR 2.1 (Invitrogen, Carlsbad, CA) following manufacturer’s guidelines. The phaI-containing plasmid was digested with BamHI and XhoI, liberating the phaI fragment with compatible cohesive ends suitable for ligation into linearized pKNOCK-KmR. The phaI-pKNOCK-KmR plasmid was introduced into Y4I via biparental mating with E. coli strain BW20767. Counterselection for transconjugants was achieved on marine basal medium (MBM) with 2 mM p-hydroxybenzoate (POB) as  the sole carbon source and kanamycin (50 µg/mL) (per liter: 1.5% (wt/vol) Sigma Sea Salts, 2.38 µM K2HPO4, 13.35 mM NH4Cl, 71 mM Tris-HCl (pH 7.5), 68 μM Fe-EDTA, trace metals and vitamins) at 30°C (19). The resulting mutant was PCR confirmed using primers phaIF and phaIR. Orientation of pKNOCK insert was confirmed using two primer sets: (i) phaIF and pKNOCK_746 and (ii) phaIR and pKNOCK_895 (Table S1). Finally, the mutant was verified by sequencing of the phaI gene and designated phaI::pKNOCK. 
Gene expression assays
RNA was isolated from cellular biomass scraped from agar (1.5%) plates and suspended in 1.5mL of β-mercaptoethanol–RLT buffer (Qiagen, Germantown, MD). The suspension was transferred to a 2.0 mL screw cap tube containing 0.2 g of low-binding 200 μm zirconium beads (OPS Diagnostics, LLC, Lebanon, NJ). The vial was then subjected to vortex mixing at 13,000 rpm for 10 minutes and incubated in the water bath (70°C) for 5 minutes. Cell debris was pelleted and lysate was transferred into a clean 1.5 mL tube. Nucleic acids were precipitated using 700 μL volume of 70% ethanol. The lysate was then transferred to a RNeasy Mini spin column and RNA was isolated following the RNeasy minikit protocol (Qiagen, Germantown, MD). Turbo DNase (Invitrogen) was used to remove DNA from samples. Reverse transcriptase (RT) was performed using Moloney murine leukemia virus (M-MLV) reverse transcriptase following manufacture’s guidelines (Invitrogen). No RT controls were included to identify any residual DNA contamination in RNA samples. Nucleic acid concentration was measured using a Nanodrop spectrophotometer (Nanodrop Technologies, Inc, Wilmington, DE). Total RNA was diluted to 6 ng/μL for use in assays. 
Quantitative reverse transcription PCR (qRT-PCR) was used to assess gene expression of igiD, phaR, pgaR, phaI, and pgaI in strains grown on agar plates for 24 hr. Position of gene disruptions and location of qPCR primer binding sites are presented in Table S2.  Amplified products ranged in size from 150 bp to 240 bp. All assays are normalized to three reference genes (rpoC, alaS, and map; Table S1). qRT-PCR data analysis and the normalized relative transcripts quantities were calculated using the qBASE method (20). 
Viable cell counts and total biomass on surface attached cultures 
To assess viability of surface attached cells, we inoculated a 96-well polystyrene plate (Costar, Corning Incorporated Corning, NY) containing a sterilized 4mm glass bead (Pyrex, Corning Incorporated Corning, NY) with Y4I variants at ~1 x 104 cells/mL and incubated at 30° C.  Y4I variants were inoculated in biological and technical triplets. A duplicate of each plate was made to assess total biomass described below. Following 20, 44, and 48 hrs, the glass bead was extracted from wells and cells were dislodged from beads as previously described (12). Briefly, beads containing cell biomass were transferred to 2 mL plastic screw cap tubes containing 940 µL of 20% YTSS (per liter: 15 g Instant Ocean, 0.8 g tryptone, 0.5 g yeast extract) and 60 µL of 10% Tween20, submerged in a sonicating water bath and sonicated for 6 minutes at 40 kHz. Samples were then vortexed for ~30 s and diluted to perform viable cell counts. 
Total biomass on surface attached cultures was assessed by performing a crystal violet stain on glass beads. Briefly, liquid was removed from wells containing glass beads. The beads were rinsed with 1.5 % sea salt solution (Instant Ocean), then extracted and moved to a clean 96-well plate containing 100 µL YTSS. Uninoculated glass beads served as a control. Crystal violet (25 µL) was added to all wells in the 96-well plate containing beads and incubated for 30 minutes at room temperature. Liquid was aspirated out of wells and stained beads were washed ~3X with 1.5% sea salt solution. Following wash, 200 µL of 95% EtOH was added to each well and allowed to sit for 1 hr to allow crystal violet to solubilize. Following solubilization, liquid was transferred to a clean 96-well plate and diluted 1:10 with 95% EtOH. Absorbance was read at 600 nm using a microplate reader (Bio Tek Instruments, Inc., Synergy HT Multi-Mode Microplate Reader, SN 270212).
Cyclic-di-GMP extraction and quantification
Cyclic-di-GMP was extracted from cell biomass in liquid and growth on agar cultures using procedure adapted from (21). For broth grown cultures, Y4I variants were grown in liquid YTSS at 30° C overnight, shaking in the dark. Cultures were reinoculated in YTSS and allowed to grow for 24 hrs under the same conditions. Following 24 hrs, 10 mL of culture was pelleted by centrifugation (3 min at 8000 rpm). Supernatant was decanted, biomass was resuspended in 1 mL 1.5% sea salt solution and transferred to a 1.5 mL microcentrifuge tube. Biomass was pelleted again, and supernatant was removed by pipetting. For surface attached cultures, Y4I variants were grown in liquid YTSS at 30° C overnight, shaking in the dark. Liquid cultures were spotted (50 µL) onto YTSS agar and incubated for 24 hrs at 30° C. Biomass was scrapped from agar using cell scrappers and transferred to a 1.5 mL microcentrifuge tube. For Y4I variants in either growth medium technical replicates were combined, if necessary, to achieve an approximate biomass of ~0.04 g. Biomass was then suspended in 100 µL extraction buffer (40% methanol, 40% acetonitrile, and 0.1 M of N formic acid) per 0.04 g of biomass. Cell slurries were then incubated at -20° C for 30 minutes. Following incubation, cells slurries were centrifuged at 4° C (13000 rpm for 3 minutes) to remove insoluble material. Lysate was neutralized with 4 µL of 15% ammonium bicarbonate per 100 µL of sample and transferred to a clean 1.5 mL microcentrifuge tube. Cyclic-di-GMP was extracted from pellet again using 50 µL of extraction buffer per 0.04 g of initial biomass. Incubated for 30 minutes at -20° C, centrifuged at 4° C at 13000 rpm for 3 minutes, and neutralized with 4 µL 15% ammonium bicarbonate and lysates were combined. Following extraction, samples were dried, and shipped for processing to the Mass Spectrometry and Metabolomics Core (Michigan State University). Samples were resuspended in ~160 µL of mobile phase A [(10 mM tributylamine +15 mM acetic acid) in 97:3/H20:MeOH]. Fluorinated c-di-GMP (Difluoro 3'3'-c-di-GMP, InVivoGen, San Diego, CA) was added as internal standard prior to being analyzed using mass spectrometry. 
Growth Inhibition assays
Growth inhibition of A. fischeri by Y4I variants was assessed as previously reported (Cude 2012, 2015). Briefly, A. fischeri was grown overnight (23°C) to an optical density (OD) of 1.5 – 2.0 at 540 nm. Cultures of A. fischeri were diluted 1000-fold and spread plated on to YTSS. Plates containing A. fischeri lawn were allowed to dry prior to spotting with Y4I variants. Y4I variants were grown to an OD of 0.6 at 540 nm and 10 µl spot plated on A. fischeri lawns and incubated at 27°C as growth rates of the two organisms are comparable at this temperature (12). Zones of clearing around Y4I strains was assessed at 48 hrs post inoculation. 
Exogenous AHL add-back assay and indigoidine quantification 
To test whether addition of exogenous AHLs could restore indigoidine production, we performed AHL add-back assays and measured indigoidine production in each Y4I variant. Y4I and variants were diluted from overnight cultures to an OD540nm of approximately 0.2. This optical density corresponds to early exponential phase in Y4I strains. Cultures were then incubated at 30° C until late exponential phase (OD540nm ~1.3) was reached. At this time, a final concentration of 13 nM 3OHC12:1-HSL was added to the liquid cultures. Technical replicates containing no added 3OHC12:1-HSL served as controls. All cultures were incubated at 30° C for approximately 16 hrs until cultures reached stationary phase (OD540nm ~2.0). Following incubation, cultures were spot plated, in triplicate, onto YTSS agar containing a final concentration of 800 nM C8-HSL. All cultures were also spot plated on YTSS agar containing no C8-HSL. Spot plates were incubated at 30°C for 24 hrs (Fig S1). Following incubation, one set of technical replicates was sacrificed to quantify indigoidine as outlined previously (12). Briefly, cells were scrapped from plates and solubilized in dimethyl sulfoxide (DMSO). To obtain purified pigment, the cells were lysed via sonication and cell debris were collect via centrifugation. Pigmented supernatant was passed through a 0.2 μm filter and transferred to a clean tube. Two microliters aliquots of the solubilized pigment were measured on a Nanodrop spectrometer (Thermo Fisher Scientific) at 612 nm. 3OHC12:1-HSL and C8-HSL were synthesized locally as described previously (14).
Statistical analysis
All data was analyzed and visualized using GraphPad Prism8 (GraphPad Software, La Jolla California USA, www.graphpad.com). Two-way ANOVA with Dunnett’s test was performed on gene expression assay data. One-way ANOVA with Tukey’s test was used to determine statistical significance in quantitative biofilm assays and cyclic-di-GMP analysis. One-way ANOVA using Dunnett’s test was used to determine statistical significance on AHL add-back assays. Mixed effects analysis was performed on viable cell counts and total biomass assays using Dunnett’s and Tukey’s, respectively, for post-hoc analyses. Outliers were identified with 95% confidence using a robust nonlinear repression model (ROUT) (22).

IV. Results
Rhodobacterales sp. Y4I possesses two QS systems: phaRI (QS1) and pgaRI (QS2). We have previously demonstrated that disruption of pgaR (QS2) results in undetectable levels of its cognate AHL, C8-HSL, which is synthesized by PgaI (14). This result indicates that the insertional mutation in pgaR leads to downstream effects on pgaI expression. Indeed, this is confirmed by the gene expression data presented in a subsequent section. In contrast, disruption of phaR (QS1) results in detectable, albeit decreased levels, of its cognate AHL, 3OHC12:1-HSL. In addition, indigoidine production is impaired, but not abolished, in this mutant (14). Thus, to fully elucidate the relative contributions of each QS system and to define the regulatory architecture governing indigoidine biosynthesis, we first generated a AHL synthase mutant in the QS1 system (phaRI) in the marine isolate, Rhodobacterales sp. Y4I. We then assessed various phenotypes of this and previously generated mutants.
The phaI mutant does not produce indigoidine and demonstrates increased biofilm formation. Unlike the phaR::Tn5-KmR (QS1) mutant, phaI::pKNOCK (QS1) does not produce detectable levels of its cognate AHL (data not shown) nor does it produce any visible levels of indigoidine, even after several days of incubation. Consistent with this phenotype, the mutant is unable to inhibit the growth of A. fischeri (Table 1). We also assessed whether insertional mutagenesis of phaI results in growth defects. Growth of phaI::pKNOCK in liquid culture was comparable to that of wildtype (Fig. S2A). Given that Y4I forms prolific biofilms and mutants in QS1, QS2, and the indigoidine synthase gene (igiD) previously demonstrated increased biofilm formation compared to wildtype, we tested biofilm formation in phaI::pKNOCK. The phaI mutant has significantly increased biofilm formation relative to wildtype as well as the other mutants assayed (Fig. S2B). 
Disruption in pgaRI (QS2) leads to a system wide decrease in QS and igiD gene expression. To examine the QS regulatory network governing indigoidine biosynthesis in Y4I, we performed gene expression assays via qRT-PCR on the following five genes: igiD (indigoidine synthase); phaR and -I (QS1); and pgaR and -I (QS2) (Fig. 1). A disruption in the luxR homolog of QS2 (pgaRI) resulted in decreased expression in all genes surveyed: a nearly 5-fold decrease in its own gene, more than 10-fold decrease in the luxI homolog of QS2, a 4-fold decrease in phaR, and a 2-fold decrease in phaI. Furthermore, this disruption in pgaR led to more than 1000-fold decrease in igiD expression (Fig. 1A). 
Insertional disruption of phaR (QS1) resulted in a 2500-fold decrease of its own expression, but only a ~3-fold decrease in expression of phaI. Expression of igiD showed a 2-fold decrease compared to wildtype (Fig. 1B). Insertional mutagenesis in phaI resulted in an 8-fold decrease in expression of its own gene and a 4-fold decrease in igiD expression (Fig. 1C). In the igiD::Tn5-KmR mutant, expression of QS1 lux homologs genes (phaRI) showed a 2-fold reduction for both genes, while expression of the luxR homolog in QS2 (pgaR) remained at wildtype levels. Insertional disruption of igiD resulted in a nearly 4-fold increase in pgaI (QS2) gene expression (Fig. 1D). 
Disruption of either QS1 gene (phaR and -I) led to greater variance in expression of the other assayed genes. For both mutants, a consistent variation is seen in the pgaI gene expression: in phaR::Tn5-KmR,  pgaI transcripts from more than 10-fold decrease to a nearly 2-fold increase in across biological replicates (Fig. 1B). In phaI::pKNOCK, pgaI transcripts range from a 5-fold decrease to a 5-fold increase across replicates (Fig. 1C). Variability within this mutant is consistent across multiple experimental assays, including biofilm formation, motility, and spontaneous pellicle formation (aka flocking) in liquid cultures (data not shown). 
Exogenous Addition of AHLs Partially Restore Indigoidine Production in QS1 Mutants.  Given AHL concentrations are decreased in all QS mutants, we next tested to see if we could chemically complement these mutants with the exogenous addition of AHLs. To mimic previously characterized AHL concentrations in Y4I during different phases of growth (14), 13 nM 3OHC12:1-HSL was added to liquid cultures during late exponential phase (OD540nm ~1.3). When cultures reached stationary phase (~16 hr following 3OHC12:1-HSL addition), they were spotted onto agar plates containing a concentration of 800 nM C8-HSL. To assess whether one AHL was driving indigoidine production, we also included 3OHC12:1-HSL and C8-HSL alone controls (Fig. S1 and Fig. 2). To determine whether exogenous addition of AHLs was able to rescue indigoidine production phenotype, we compared the absorbance of our AHL treated mutants to that of the no AHL control in our indigoidine null mutant (igiD::Tn5-KmR) (Fig. 2). To assess significance in rescuing indigoidine production, we compared AHL treatments to no AHL treatments within mutant genotypes (Fig. 2). 
Exogenous addition of AHLs did not produce any significant response in indigoidine production in wildtype, the indigoidine null mutant or the pgaR (QS2) mutant (Fig. 2A, 2B and 2E). Addition of 3OHC12:1-HSL (QS1) alone does not significantly restore pigment production in the phaR mutant, providing additional support for the non-functionality of PhaR in that strain (Fig 2C). Both phaI and -R (QS1) mutants show some restoration of indigoidine production when supplied with exogenous C8-HSL.   When supplied with either AHL, but not both, the phaI mutant shows significantly increased indigoidine production. Interestingly, C8-HSL addition restores indigoidine production to 33% that of wildtype in the phaI mutant compared to the chemical complement of its own AHL (3OHC12:1-HSL) which restored production to 22% that of wildtype. Addition of both AHLs in combination resulted in less than 10% wildtype levels of indigoidine production in this mutant background (Fig 2D). 
Temporal Analysis Reveals Potential Role for QS in Biofilm Regulation. We next assessed whether alterations in the QS pathways also lead to differences in rates of surface colonization and biofilm formation.  The indigoidine biosynthesis mutant was included for reference in these assays. Quantification of viable surface attached cells over a 68-hr period revealed all mutants were impaired in their ability to either attach or grow on the surface during the initial growth phase (20 hr). However, those differences were insignificant during later sampling efforts (Fig 3A). In contrast, quantification of biofilm biomass showed the opposite trend. All mutants were indistinguishable from wildtype at the 20 hr timepoint, but variation between and among wildtype and the mutants became pronounced with time. For example, both phaR (QS1) and pgaR (QS2) mutants show significantly elevated biofilm biomass relative to wildtype at 44 hr, but these biofilms were not significantly different by the final time point (68 hr). Conversely, phaI (QS1) mutant was indistinguishable from wildtype until the final time point, at which point it was 32-42% greater than wildtype and all other variants (Fig 3B). 
Cyclic-di-GMP is higher in surface attached cultures and influenced by QS. In many biofilm forming bacteria the global secondary messenger, c-di-GMP, is integrated into the regulatory network dictating the transition between motile and sessile lifestyles (5). Thus, we wanted to investigate intracellular c-di-GMP concentrations in Y4I and the panel of QS and indigoidine mutants. C-di-GMP concentrations are significantly increased in agar grown Y4I cultures relative to broth cultures (Fig. 4A). Furthermore, all mutants have significantly reduced c-di-GMP concentrations, ranging from 15-33% of wildtype levels when strains are grown in liquid culture and 4-23% of wildtype when grown on agar surfaces (Fig 4B-C). 

V. Discussion 
AHL-based QS networks are prevalent in members of the Roseobacter clade where they regulate physiologies predicted to be central to the ecological success of group members (11, 23). The high incidence of multiple QS systems in individual strains presents opportunity for different QS network architectures and signaling circuities to have evolved, the extent of which has not been fully evaluated for roseobacters. From studies of diverse microbes, three defined QS network architectures have been proposed and provide a valuable comparative framework: (i) ‘One-to-One’ system, in which a single receptor responding to a single signal controls the entire QS response; (ii) ‘Many-to-One’ parallel circuit, in which information contained in multiple autoinducers are integrated together to control the QS response; and (iii) ‘Many-to-One’ hierarchical system, in which many QS receptors are connected in a signaling cascade (24). Of the handful of Roseobacters that have been genetically characterized with regards to QS, two appear to employ a hierarchal regulatory circuit: Ruegeria mobilis KLH11 (25) and Dinoroseobacter shibae (26). A third, Phaeobacter inhibens, appears to employ a parallel regulatory circuit to regulate the production of the antimicrobial trophodithietic acid (27). Here, we propose that Y4I uses a ‘Many-to-One’ hierarchal QS circuit to mediate indigoidine production and biofilm formation. We also provide evidence for integration of a global regulatory metabolite (c-di-GMP) into this QS circuitry. 
Several lines of evidence suggest that QS2 (pgaRI) is at the top of the hierarchical regulatory network that integrates input from QS1 to govern indigoidine biosynthesis. Expression of both QS systems and the indigoidine synthase gene, igiD, is negatively influenced when the QS2 transcriptional regulator (pgaR) is disrupted. This mutant is also null for its cognate AHL. In contrast, disruption of either QS1 component (phaR or -I) has no significant effect on gene expression of QS2 components, though pgaI expression shows considerable and consistent variation across biological replicates in both QS1 mutants.  We postulate that the variation in pgaI expression is a manifestation of global scale misregulation resulting in some degree of stochasticity within these mutants. This suggests a role for the QS1 system in regulatory homeostasis in this strain. Generation and study of a pgaI mutant would further support this finding.
The phenotypic response of the mutants to exogenous AHL additions provide further evidence for the predominant role of QS2 as well as cross-signaling between the two QS systems. With regards to indigoidine production, the QS2 mutant (pgaR) is unresponsive to any of the AHL additions. However, both QS1 mutants can be partially restored for this phenotype. When supplied with exogenous C8-HSL (QS2), indigoidine production is evident in both QS1 (phaRI) mutants. Addition of its own cognate AHL (3OHC12:1-HSL) results in partial restoration of indigoidine in the phaI mutant. However, addition of both AHLs does not restore pigment production. That both AHLs in combination are unable to chemically complement indigoidine production is intriguing. One explanation for this result could lie in the promiscuous nature of AHLs and their receptors. Alternations in the length and degree of saturation in acyl chain structures impact binding affinity of the transcriptional regulator, influencing the genetic response (28, 29).  Simultaneous addition of both AHLs may oversaturate the system, resulting in promiscuous binding of the transcriptional regulators and disruption of the regulatory network. Indeed, addition of AHL structural analogs is a proposed mechanism for combating bacterial pathogens reliant on QS for virulence (30).  Genetic complementation of these mutants would enhance confidence in the results and help elucidate the mechanism underlying the results presented here.
	The two QS mutants exhibit temporal differences regarding biofilm formation, indicating a role for this regulatory network in surface colonization and biofilm dynamics in Y4I.  This finding is consistent with studies in other bacteria where QS can influence all stages of biofilm progression: from attachment and recruitment to maturation and dispersal (31, 32). By monitoring Y4I biofilm formation over the course ~70 hours, the cyclical nature of this growth modality is evident. Comparison between and amongst mutant and wildtype strains reveals a degree of asynchronicity in the biofilm mass of QS mutants, supporting a role for QS in mediating periodic biofilm dispersal.  QS-regulated biofilm dispersion has been proposed to prevent overcrowding in the sponge symbiont R. mobilis (11). Furthermore, induction of biofilm dispersal via QS in P. inhibins is thought to contribute to the switch between mutualism (attachment) and pathogenesis (dispersion) with its host (27).  The production of the Y4I QS1 signal (3OHC12:1-HSL) is biphasic across the growth cycle, highest during lag and late stationary phases of growth. A previous interpretation of these data was that this AHL represents an important signal when cell growth is relatively static and, thus, may impart information on metabolic status (14). This interpretation combined with data presented here suggests a role for cellular metabolic status in biofilm dynamics, particularly dispersion. Indeed, this topic has recently gained attention for pathogenic microbes (33).  
C-di-GMP is integrated into QS network
The global secondary messenger, c-di-GMP, relays information in response to metabolic state and environmental cues. This metabolite has been shown to modulate biofilm dispersal in many bacteria (33, 34). Consistent with the  general paradigm (5),  intracellular c-di-GMP concentrations in Y4I correlate with growth modality: they are elevated in surface-grown cultures relative to liquid cultures. C-di-GMP concentrations are not only altered in Y4I strains with disruptions in either QS system but also the indigoidine biosynthesis mutant, suggestive of a direct link between these secondary metabolites. In fact, glutamate has been shown to play a key role in  biofilm formation by regulating intercellular pools of c-di-GMP in P. aeruginosa (35, 36). In Y4I, glutamate is an essential precursor for indigoidine (12, 37, 38). Thus, the integration of c-di-GMP and QS signaling pathways may provide an avenue for convergence of information on cellular status from different central metabolic pathways in Y4I.  
Integration of c-di-GMP with bacterial regulatory signaling pathways is not uncommon. For example, in several Alphaproteobacteria, c-di-GMP contributes to the regulation of the biphasic swim-stick switch controlled by the CtrA phosphorelay “master regulator” system (26, 39–42). It is plausible that c-di-GMP and the CtrA phosphorelay system are also integrated into the QS regulatory network architecture governing indigoidine biosynthesis in Y4I.  Indeed, we have found a putative DNA binding half-site for the master regulator, CtrA, located in the QS2 (pgaRI) operon (Fig S3). Furthermore, integration of the CtrA and QS has been demonstrated in other roseobacters. In R. mobilis, QS regulates the lifestyle switch from motile (swim) to sessile (stick) by controlling the expression of CtrA (43). Previous studies have shown that c-di-GMP is also integrated into the QS of D. shibae (44) and regulates the expression of ctrA in a related alphaproteobacterium, Rhodobacter capsulatus (45). Thus, integration of these central signaling pathways may be a mechanism utilized by members of the Roseobacter clade to aid in their colonization success. 
	In an effort to better contextualize the regulatory network architecture that may govern these key Roseobacter physiologies in marine ecosystems, we present a working model that integrates the key players discussed here (Fig. 5). Implicit in this model is a connection between metabolism and physiology. Quorum sensing molecules are dependent on metabolic precursors  (46). Additionally, c-di-GMP, a key intermediate linked to metabolic state has been demonstrated to regulate QS, either directly or indirectly, in Alphaproteobacteria (31, 41, 47). We postulate this mechanism of regulation applies to the QS networks in Y4I. We also hypothesize a role for the CtrA phosphorelay system in regulating surface growth associated physiologies, including attachment, mechanosensing, flagella and biofilm formation in Y4I (48). We anticipate future studies will more fully elucidate the extent and nature of c-di-GMP and CtrA to antimicrobial production and biofilm in this strain. 
Ecological Context of QS Signaling Dynamics
QS has been shown to contribute to competitive physiologies, such as biofilm formation and antimicrobial production, in members of the Roseobacter clade (e.g.(27, 49, 50)and reviewed in Zan et al., 2014). Here, we provide evidence that Y4I employs a hierarchical QS circuit to mediate biofilm formation and indigoidine production. At the top of the hierarchical network in Y4I is one of the most common marine signaling molecules: C8-HSL (12, 51–55). It is intriguing that this dominant AHL, which is produced by a number of aquatic microorganisms, including A. fischeri and A. anguillarum, is also required for indigoidine biosynthesis in Y4I, which inhibits the growth of both of these species (14, 56, 57). Given the universality of C8-HSL, the exploitation of this ubiquitous signaling molecule by Y4I may be central to its successful competitive strategies. 



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Appendix: Tables

  
Table 2.1 Y4I variant phenotypes

	
	WT
	pgaR::Tn5-KmR
	phaR::Tn5-KmR
	phaI::pKNOCK
	igiD::Tn5-KmR

	Indigoidine Productiona
	+
	-
	+/-
	-
	-

	Growth Inhibitionb 
	+
	-
	-
	-
	-



aIndigoidine production was rated by appearance of coloration of colonies following inoculation at 48 hrs (+), within 5 days (+/-), or not at all (-) during incubation. 
bGrowth inhibition of Aliivibrio fischeri was either (+) if a zone of clearing was present or (-) if there was no zone of clearing.

Appendix: Figures

Figure 2.1 Gene expression of luxRI homologs and igiD synthase from agar grown cultures. Relative gene expression for pgaR::Tn5-KmR (A), phaR::Tn5-KmR (B), phaI::pKNOCK (C), igiD::Tn5-KmR (D). Gene expression was normalized to three housekeeping genes (rpoC, alaS, and map) and expressed relative to WT, set to 1. Each data point represents the average of three technical replicates and errors bars represent standard deviations between three biological replicates. Statistical significance was calculated using a two-way ANOVA (p < 0.05 [*], 0.01 [**], 0.001 [***], 0.0001 [****]).


Figure 2.2 Indigoidine production in response to growth-phase dependent AHL exogenous addition. Indigoidine quantification in Y4I variants: WT (A), phaR::Tn5-KMR (B), and phaI::pKNOCK (C) following exogenous addition of AHLs, denoted as C8-HSL (Green), 3OHC12:1-HSL (Orange), Both AHLs (Blue), and no AHLs (Gray). Indigoidine quantification was normalized to biomass and compared to no AHL control in the igiD::Tn5-KmR mutant (red dotted line). Error bars represent standard deviations from the mean of three biological replicates. Statistical significance was calculated by comparing AHL treatments to the no AHL control within strain genotypes. Asterisks represent statistical difference (p < 0.01). Data for the pgaR and igiD mutants can be found in Figure S3.


 
Figure 2.3 Surface Colonization and Biofilm production over time in Y4I mutants. Viable cell counts of surface attached cultures in Y4I variants (A). Each data point represents the average of three biological and three technical replicates. Data was normalized to cell abundance in liquid culture at the time of inoculation. Error bars represent the standard error of the mean of biological and technical triplicates. The asterisks denote statistical significance from WT at that time point (P < 0.05 [*], 0.01 [**], 0.001 [***]). Total biofilm of surface attached cultures in Y4I variants as determined by crystal violet assay (B). Data is representative of the mean of three biological and three technical replicates. Letters that are not shared represent significant difference (p < 0.05). Error bars represent the standard deviation from the mean.  





Figure 2.4 Cyclic-di-GMP concentrations in broth and agar grown cultures. A comparison of intracellular cyclic-di-GMP concentration in WT cultures grown on agar and in broth culture (A). Intracellular c-di-GMP concentration of Y4I variants grown on broth (B) and in agar culture (C) are shown.  Each data point represents the average of three technical replicates. Error bars represent the standard deviation of the mean. Asterisks represent statistical significance (p < 0.05 [*], 0.001 [***], 0.0001 [****]).  




Figure 2.5 Working model of the regulatory network governing key physiologies in strain Y4I. Left panel shows genetic regulation during growth at low cell densities when Y4I is growing planktonically. During this growth modality, 3OHC12:1-HSL (QS1) concentrations are highest and we hypothesize that surface growth associated genes (e.g., mechanosensing, attachment, biofilm formation, flagella biosynthesis, and/or ctrA phosphorelay) are repressed, either directly or indirectly through QS1. Right panel shows genetic regulation during growth on a surface. It is predicted that when concentrations of C8-HSL are highest, QS2 represses expression of QS1, either directly or indirectly, allowing for the transcription of surface growth associated genes. Both c-di-GMP and CtrA are expected to play a role in activating surface-growth associated genes, though the specific mechanism of this regulation is currently unknown. Black lines are indicative of activation while red represents inhibition. Solid lines represent known pathways, while dotted lines are hypothesized.  


Appendix: Supplemental Materials
Table S 2.1. Table of primers. Primers used in this study for gene expression analysis and construction of phaI mutant.  Primer sets are listed together, sharing a prefix and designated with either “for” or “F” to indicate forward primer or “rev” or “R” to indicate reverse primer.
	Primer Name
	Sequence
	TM
	Product size
	Source

	map_for
	GTG TTC CAC GCC CCG CCC AAC
	70.4
	
	Cude et al., 2012

	map_rev
	CCC GGC CGG TGA CAG GGT GAA
	70.4
	200bp
	Cude et al., 2012

	rpoC_for
	CGG CGC TGA AGC GAT CCG TGA
	68.4
	
	Cude et al., 2012

	rpoC_rev
	CCG GAC GGT TGC CCG ATT CCA
	68.4
	200bp
	Cude et al., 2012

	alaS_for
	GCT GTG GGC GGA GGG GCA ATG
	70.4
	
	Cude et al., 2012

	alaS_rev
	GCC GAT CGA ACC GCC GGT GAC
	70.4
	200bp
	Cude et al., 2012

		Y4I_3464_for



	GGC AAG ATC CAG GGC ATA C
	60.44
	
	This study

		Y4I_3464_rev



	ATC AGG AAC GAC CGG TAC TC
	59.02
	302bp
	This study

	phaI_qPCR_for1
	CAG CCT TGG CGC CTC GGA AA
	66.6
	
	Cude et al., 2015

	phaI_qPCR_rev1
	TGC AGC GGA GCG AAT TGC GA
	68.6
	216bp
	Cude et al., 2015

	pgaI_qPCR_for1
	GGC GGC TCC ATG CGG TTT CT
	66.6
	
	Cude et al., 2015

	pgaI_qPCR_rev1
	CCT GCT GCG ATC CTG CCC TG
	64.5
	155bp
	Cude et al., 2012

	qPCR_igiD_for
	GGT CAG AAA GGA CGC GTC GCG G
	70.1
	
	Cude et al., 2012

	qPCR_igiD_rev
	AGC GCG CGA TGC CGA GCT GAT C
	70.1
	229bp
	Cude et al., 2012

	pgaR_qPCR_for1
	ATC AGG GGT CCG AAC GGC CA
	66.6
	
	This study

	pgaR_qPCR_rev1
	CGG CTG TAG CCG ATC GCC AG
	68.6
	227bp
	This study

	phaR_qPCR_for1
	GGT GGG CTT TGA CGG GGT CG
	68.6
	
	This study

	phaR_qPCR_rev1
	AGC CGC TCC AGC TCC TTC CA
	66.6
	159bp
	This study

	phaI_F
	CGC AAG CAG AAG CTG GTG GA
	64.5
	
	This study

	phaI_R
	ATA CCC GAA CCG CCT CAG CA
	64.5
	429bp
	This study

	pKNOCK_746

	CAC TTA ACG GCT GAC ATG G

	52.6
	
	This study

	pKNOCK_895

	TTA ATT CGA CGC GTC CTC

	50.0
	149bp
	This study




2



Table S 2.2. Position and nature of insertional mutations and location of qPCR primers.
	Mutant designation
	Gene disrupted and type of insertion
	Length of wildtype gene (bp)
	Location of insertiona
	qPCR primer locationa,b
	length of insertion (bp)
	Source

	
	
	
	
	
	
	

	Y401BD4
	igiD::Tn5-KmR
	3942
	595
	3288 (F) 3516 (R)
	4080
	Cude et al., 2012

	Y411CE4
	pgaR::Tn5-KmR
	720
	269
	354 (F) 580 (R)
	4080
	Cude et al., 2015

	Y412AE6
	phaR::Tn5-KmR
	636
	45
	90 (F)
248 (R)
	4080
	Cude et al., 2015

	phaI::pKNOCK
	phaI::pKNOCK
	666
	268
	829 (F) 1044 (R)
	2400
	This study


 










a Nucleotide position within the designated gene
b(F) = position of start of forward primer and (R) = position of start of reverse primer 
Figure S 2.1 Overview of methodology for AHL add back. Overnight cultures (3 biological and 3 technical) of Y4I and variants were split into duplicates (now 30 cultures) and diluted to early exponential phase (OD540 ~ 0.1 nm) and allowed to grow until they reached mid-exponential phase (OD540 ~ 1.0 nm). 3OHC12:1-HSL was added to one set of the duplicate cultures (red arrow). The other set of duplicate cultures became the C8-HSL alone treatment and the no AHL control.  All cultures were allowed to continue growing until 24 hrs had been reached. All cultures were then spotted in 10 µL spots onto 10 mL YTSS agar plates with or without C8-HSL and incubated for 24 hrs. The resulting 4 conditions were then analyzed for their indigoidine production for each Y4I variant: both AHLs (blue), 3OHC12:1-HSL alone (yellow), C8-HSL alone (green), and no AHLs (gray). 



Figure S 2.2. Growth dynamics in liquid culture (A) and biofilm formation (B) in Y4I and mutants. For liquid cultures, data points are representative of the average of biological quadruplets. Biofilm formation was assessed by crystal violet assay at 24 hrs post inoculation. Data bars are representative of the average of two biological replicates containing technical septuplets. Error bars represent standard deviation from the mean and statistical significance was calculated using a one-way ANOVA. 


Figure S 2.3. Half CtrA binding site within pgaRI (green) operon. pgaRI (RBY4I_1689, RBY4I_3631) and phaRI (RBY4I_1027 and RBY4I_3464)  genes were queried for CtrA binding motifs based on the approach outlined in Laub et al. 2000 (Laub et al., 2000). A CtrA half binding site within the pgaRI cassette was found (sequence shown below in box) starting at ~484 bp. Genes are represented by page arrows and shown to scale. Black arrows represents genes immediately downstream of QS2 (pgaRI). 









Methodology

CtrA Motif Identification. The cell cycle regulator, CtrA, is conserved among Alphaproteobacteria (Brilli et al., 2010). We searched for both full and half CtrA binding sites in the upstream regions of phaRI and pgaRI using consensus sequences as determined by Laub et. Al, 2001 (Laub et al., 2000). We found a half CtrA binding site with high similarity in the intergenic region between pgaR and pgaI (Fig. S3). Indicating that CtrA may be integrated into this QS circuitry. 



Secondary Metabolite Production in Synthetic Roseobacter Communities orchestrates Biofilm dynamics

A version of this chapter was written by April C. Armes, Jillian L. Walton, and Alison Buchan and is intended to be submitted for publication.
	

A.C.A. and A. B. conceived and designed the experiments for surface attachment and biofilm formation assays. A.C.A. and J. L. W. conducted the experiments, carried out data analysis, generated figures, and wrote the manuscript. The manuscript was edited by A.C.A, J. L. W., and A. B.  

I. Abstract
Within marine ecosystems, biofilms are critical to ecosystem functions contributing to processes such as global biogeochemical cycling, bioremediation, and biofouling. Biofilms are a consortium of microorganisms growing on a surface, suspended in a self-produced polymeric matrix. The physical matrix and spatial arrangement of cells within a biofilm facilitates a myriad of interactions, both cooperative and competitive in nature, between biofilm members. These interactions are often mediated by quorum sensing (QS), signaling systems that elicit population-level responses in a cell density dependent manner, including the expression of secondary metabolites with antimicrobial activity. Despite the crucial role of secondary metabolites in biofilm dynamics, few studies have directly explored this role. One area of interest is the influence of primary surface colonizers on interactions within a biofilm. Members of the Roseobacter clade, a group of marine heterotrophic bacteria, are primary and aggressive surface colonizers and represent a large portion of microbial members in coastal environments making clade members excellent models to study community interactions in marine ecosystems. Given their diverse metabolic capabilities and numerous QS pathways, we utilized a five-member synthetic community of Roseobacter members to address whether secondary metabolite production, mediated by QS, contributes to microbial community structure by introducing QS mutants to a synthetic biofilm community in the presence of complex and defined media. Our findings indicate that a common marine QS signaling molecule, N-octanoyl-L-homoserine lactone (C8-HSL), influences community structure in a biofilm provided grown on a complex medium (i.e., tryptone and yeast extract). When given a more recalcitrant and defined carbon source (i.e., p-coumaric acid) the abundance in community structure by the primary surface colonizer, Rhodobacterales strain Y4I is delayed. Furthermore, our data suggests that an inactivation of QS systems in Y4I, results in increased community cooperation. These data provide evidence of the role a primary surface colonizer, such as Y4I, plays in biofilm community structure and dynamics in marine ecosystems.


II. Introduction

Microbial interactions play critical roles in defining microbial community structure and function. These microbial interactions span from more cooperative interactions, such as resistance to antimicrobials and co-metabolism (Burmolle 2006, Leriche et al 2003, and Pande 2015), to more competitive interactions, such as production of inhibitory compounds and competition for resources (Rao 2005, Cude 2012, and Manfield et al 2001). Many types of microbial interactions are mediated by small molecules that are not essential to primary metabolism but are produced to facilitate interactions between microbes and their biotic and abiotic environments (i.e., secondary metabolites)(Miller et al., 2001). Two common classes of secondary metabolites produced by microbes are signaling molecules and antimicrobial compounds. A well-known group of small, diffusible signaling molecules, the acylated homoserine lactones (AHLs), are common to many Proteobacteria where they are used to coordinate gene expression in a population density dependent manner (i.e., quorum sensing (QS)). Canonical AHL-mediated QS consists of a two-component system consisting of a transcriptional regulator (LuxR-type protein) and an AHL synthase (LuxI-type protein) that produces the diffusible ligand. When bound, these protein-ligand complexes can elicit global changes in gene expression (Miller et al., 2001). Common bacterial traits that are QS-regulated include but are not limited to: bioluminescence, pathogenicity, biofilm formation, and antimicrobial production (citation for each -- Case et al, 2008; Wagner-Dobler et al, 2005; Duerkop et al 2020). Antimicrobials are thought to contribute to microbial interactions principally through inhibition of the growth of competitors. However, for some microbes, these compounds may themselves function as inter-microbial signals at low concentrations (Beyersmann et al 2017). Thus, these two classes of molecules are crucial to competitive and cooperative microbial interactions.
In many environments, diverse microorganisms are enclosed in biofilms where they are in close physical association with one another and encased in a self-produced polymeric matrix. The biological and physicochemical properties of biofilms make them ideal systems to study microbial interactions, especially those that are mediated by diffusible small molecules. In addition, biofilms play critical roles in ecosystem functioning, where they mediate transformations key to biogeochemical cycling (Decho 2017), bioremediation (Yoshida 2009), and biofouling (Adnan 2018). In turn, biofilm-associated microorganisms are afforded some degree of protection by the biofilm matrix from external environmental stressors, predators, toxins, and antibiotics (Dang et al., 2016). Secondary metabolites commonly have enhanced biosynthesis in biofilms, leading to higher concentrations of these compounds in the local environment and subsequently influencing microbial interactions along the spectrum from cooperation to competition (Rieusset et al. 2020).
Current understanding of QS-mediated microbial interactions within biofilms is principally based on co-culture and natural mesocosm assemblages (Chandler et al. 2012; Dobretsov et al. 2011). While valuable, co-culture studies can be limited by an over-simplification of microbial interactions. On the other hand, mesocosm experiments yield a wide range of variables to consider when trying to tease apart microbial interactions (Smalley et al 2015; Smith at el 2017 & Agogué et al. 2014). Synthetic intentional communities provide an opportunity to limit community complexity while still allowing for some of the original interactions to remain and have been used recently in surface colonization and biofilm studies to mimic interactions in natural ecosystems (Cude et al 2012; Ren et al 2015; Quigley 2019; Correa de Souza et al 2020). Despite the recent emergence of multispecies-based biofilm studies, our knowledge of microbial interactions within natural biofilm communities is still relatively incomplete, specifically those cooperative and competitive interactions influenced by QS. A critical aspect to understanding these QS-linked interactions within multispecies biofilm communities is to evaluate the fitness, physiological, and ecological aspects between individual species and a multispecies biofilm consortium. To address this knowledge gap, here we systematically evaluated the contribution of QS using a series of bacterial mutants unable to produce secondary metabolites in a low diversity and defined synthetic community to investigate cooperation and competition within a biofilm.  
Here, we used a synthetic five-member intentional community composed of members of the ecologically relevant Rhodobacterales order. Members of this heterotrophic clade of bacteria, known as the Roseobacter clade, comprise up to 20% of the microbial communities in marine ecosystems, possess a large genetic repertoire, and high metabolic diversity. As a result of their vast metabolic capabilities, Roseobacters are adept at transforming phenolic compounds and play a role in carbon and sulfur cycling (Wagner-Dobler and Biebl, 2006; Buchan, et al. 2019). In marine ecosystems Roseobacters are important primary surface colonizers, promoting surface colonization and biofilm formation of other marine bacteria through the production of secondary metabolites (Kviatkovski, et al., 2015). In addition to the colonization role of secondary metabolites, growth substrate also has been reported to play a role in defining Roseobacter synthetic communities (Quigley et al., 2019). Due to their predilection to form a biofilm on a variety of surfaces and robust secondary metabolite production, members of the Roseobacter clade are ideal model organisms for examining the molecular mechanisms that mediate the cooperative and competitive microbial interactions within biofilm communities.
III. Materials and Methods
Strains, growth conditions, and maintenance
Four of the strains used in this study Rhodobacterales strain Y4I, Sagittula stellata sp. E-37, Citreicella sp. SE45, Sulfitobacter sp. EE-36 were previously isolated from southeastern United States estuaries or coastal waters (Buchan et al., 2000, 2004). Roseovarius nubinhibens ISM was previously isolated from the Caribbean Sea (González et al., 2003). Previously established Y4I mutants: igiD::Tn5-KmR, pgaR::Tn5-KmR, phaR::Tn5-KmR, and clpA::Tn5-KmR were generated using a mini Tn5 transposon system (Cude et al., 2012, 2015). The phaI::pKNOCK mutant was previously generated using targeted insertional mutagenesis (Armes and Buchan, 2021). Due to the nature of mutagenesis, all Y4I mutant variants carry a kanamycin resistance gene.
All strains were maintained on YTSS agar [per liter: 15 g Instant Ocean (Thermo Fisher Scientific), 15 g agar (Thermo Fisher Scientific), 4 g tryptone, 2.5 g yeast extract].