Quorum sensing and host killing in an insect symbiosis

Sodalis praecaptivus is a recently isolated, novel bacterium that was isolated from a wound on the hand of a 71-year-old human patient. Phylogenetic analysis and comparative genomics revealed that this organism is closely related to members of the Sodalis-allied clade of insect endosymbiotic bacteria. This thesis deals with two investigations of this bacterium. The first is a study providing a physiological and biochemical characterization of this organism. The second is an analysis of the relationship between quorum sensing, virulence modulation against an insect host and a novel self-regulatory population control phenomenon in S. praecaptivus. It is proposed that using a population density signal to modulate virulence and growth could allow this organism to balance the need for host invasion against the need to maintain an asymptomatic infection of high density within an insect vector. This would allow the insect vector to transmit S. praecaptivus between potential plant and animal hosts without significant loss of fitness and might be a crucial step in the conversion of free living antecedent bacteria to mutualistic symbionts.

QUORUM SENSING AND HOST KILLING IN AN INSECT SYMBIOSIS by Abhishek Chari A thesis submitted to the faculty of The University of Utah in partial fulfillment of the requirements for the degree of Master of Science Department of Biology The University of Utah May 2017 Copyright © Abhishek Chari 2017 All Rights Reserved The University of Utah Graduate School STATEMENT OF THESIS APPROVAL The thesis of Abhishek Chari has been approved by the following supervisory committee members: Colin Dale , Chair 12/10/2015 Date Approved David Blair , Member 12/10/2015 Date Approved Jon Seger , Member 12/10/2015 Date Approved and by Denise Dearing the Department of , Chair of Biology and by David B. Kieda, Dean of The Graduate School. ii ABSTRACT Sodalis praecaptivus is a recently isolated, novel bacterium that was isolated from a wound on the hand of a 71-year-old human patient. Phylogenetic analysis and comparative genomics revealed that this organism is closely related to members of the Sodalis-allied clade of insect endosymbiotic bacteria. This thesis deals with two investigations of this bacterium. The first is a study providing a physiological and biochemical characterization of this organism. The second is an analysis of the relationship between quorum sensing, virulence modulation against an insect host and a novel self-regulatory population control phenomenon in S. praecaptivus. It is proposed that using a population density signal to modulate virulence and growth could allow this organism to balance the need for host invasion against the need to maintain an asymptomatic infection of high density within an insect vector. This would allow the insect vector to transmit S. praecaptivus between potential plant and animal hosts without significant loss of fitness and might be a crucial step in the conversion of free living antecedent bacteria to mutualistic symbionts. iii TABLE OF CONTENTS ABSTRACT…………………………………………………………………………………………………iii LIST OF TABLES…………………………………………………………………………………………..v LIST OF FIGURES………………………………………………………………………………………...vi ACKNOWLEDGEMENTS………………………………………………………………………………...vii 1. INTRODUCTION………………………………………………………………………………………...1 1.1 References…………………………………………………………………………………....2 2. PHENOTYPIC CHARACTERIZATION OF SODALIS PRAECAPTIVUS SP. NOV., A CLOSE NON-INSECT-ASSOCIATED MEMBER OF THE SODALIS ALLIED LINEAGE OF INSECT ENDOSYMBIONTS………………………………………………………………………………………...3 2.1 Description of Sodalis praecaptivus sp. nov………………………………………………8 2.2 Acknowledgements…………………………………………………………………………..8 2.3 References……………………………………………………………………………..……..8 2.4 Supplementary Section……………………………………………………………...……..10 2.4.1 List S1: M9 Media……………………………………………………………….11 3. AN INSECT KILLING PHENOTYPE IN SODALIS PRAECAPTIVUS QUORUM SENSING MUTATIONS………………………………………………………………………………………………13 3.1 Abstract…………………………………………………………………………………..…..14 3.2 Introduction……………………………………………………………………...…………..14 3.3 Results……………………………………………………………………………………….16 3.4 Discussion…………………………………………………………………………….……..24 3.5 Materials and Methods……………………………………………………………………..28 3.6 Acknowledgements……………………………………………………………………..…..32 3.7 References…………………………………………………………………………………..70 4. CONCLUSION……………………………………………..…………………………………………..74 iv iv LIST OF TABLES 2.1. Comparison of carbon source fermentation………………………….………………………….....8 2.S1. API 50 CH…………………………………………………………………………………………..10 3.1. OHHL dependent transcriptomes of ΔypeI mutant strain………………..................................34 3.2. Transcriptomic comparison of ΔyenR and ΔypeR mutant strain………………………………48 3.3. Pairwise log-rank tests on weevil survival ………………………….…………………………….61 3.4. Weevil infection experiments with S. praecaptivus mutant strains……………………………..65 v v LIST OF FIGURES T 2.1. Phylogeny of strain HS and related Sodalis-allied endosymbionts and free living bacteria……………………………………………………………………………………….…………......6 2.2. MALDI-TOF MS dendrogram derived from comparison of the Sodalis praecaptivus sp. nov. profile……………………………………………………..…....................................................7 2.S1. Swarming motility of Sodalis praecaptivus sp. nov……………………………....…………….12 3.1. Characterization of the S. praecaptivus QS System……………………….…………………….33 3.2. Transcriptomic (RNA-Seq) analysis of genes mediating QS in S. praecaptivus………..........47 3.3. Weevil movement following injection of S. praecaptivus WT and ΔypeI strains……….……..63 3.4. Weevil survival following injection of WT and mutant S. praecaptivus…………….................64 3.5. QS induces growth suppression in S. praecaptivus…………………………….……………….67 3.6 Inactivation of cpmA relieves growth suppression in S. praecaptivus………….…..................68 3.7 Growth curves.……………………………………………………....................…………………...69 3.8. Bacterial infection densities following microinjection.……………..…….………………………70 vi vi ACKNOWLEDGEMENTS I would like to thank all the members of the Dale Lab, and the members of my committee for all their help and support during my time at the University of Utah. viivii 1 1. INTRODUCTION Bacteria that are obligately associated with their host are seen to have a reduced genome size and a reduced gene set that is compatible with the symbiotic lifestyle (1). The process of genome degeneration in obligate symbionts has been studied in great detail but the issue of how these obligate, mutualistic associations arose in the first place is still unresolved. Any organism that tries to colonize an insect must be able to do two things: evade or repel the host insect's immune response and be able to grow within the insect to maintain a continuous but asymptomatic infection. These two strategies will allow the bacteria to maintain the host or vector long enough to be able to transmit itself to other organisms that the vector comes into contact with (2). Sodalis praecaptivus, a novel bacterium isolated from a human patient, has been shown to be closely related to members of the Sodalis-allied clade of insect endosymbionts. This clade of endosymbiont bacteria is found in a wide range of insect hosts (3). Different mechanisms have been proposed for this spread of symbionts among such a wide range of insects, including the presence of environmental progenitor bacteria. Analyses comparing the genome sequences of S. praecaptivus and related insect symbionts highlight the possibility that close relatives of S. praecaptivus gave rise to mutualistic symbionts in a range of insect hosts (2). Chapter 1 focuses on the biochemical and phylogenetic analysis of S. praecaptivus, on the basis of which this organism was given formal scientific nomenclature. The tests shown in Chapter 1 also demonstrated that the organism has a wide repertoire of metabolic properties, which is consistent with the presence of a relatively large gene inventory (when compared to other members of the Sodalis-allied clade). S. praecaptivus was shown to be the first representative of the Sodalisallied clade of bacteria that has sufficient metabolic capabilities to sustain growth in minimal media. Chapter 2 focuses on the quorum sensing pathway that exists in S. praecaptivus. Mutants that lack either quorum signal synthesis or sensing ability were shown to kill grain 2 weevils that they were introduced into, in contrast to S. praecaptivus wild type bacteria that maintained an asymptomatic infection in them. S. praecaptivus was also shown to suppress its own growth under the influence of its quorum signal molecule. Taken together, these two chapters show the range of capabilities that S. praecaptivus has to exist. It exists as a free-living organism while also being able to colonize an insect host without significantly affecting the host's fitness. It is hypothesized a commensal relationship between the bacterium and the insect would allow the insect to vector S. praecaptivus between potential animal and plant hosts. The ability of S. praecaptivus to not harm its host might also contribute to the widespread presence of the Sodalis-allied clade of bacteria in insects. 1.1 References 1. McCutcheon JP, von Dohlen CD. An interdependent metabolic patchwork in the nested symbiosis of mealybugs. Curr Biol. 2011;21(16):1366-72. 2. Clayton AL, Oakeson KF, Gutin M, Pontes A, Dunn DM, von Niederhausern AC, et al. A novel human-infection-derived bacterium provides insights into the evolutionary origins of mutualistic insect-bacterial symbioses. PLoS Genet. 2012;8(11):e1002990. 3. Snyder AK, McMillen CM, Wallenhorst P, Rio RV. The phylogeny of Sodalis-like symbionts are reconstrcuted using surface-encoding loci. FEMS Microbiol Lett. 2011;317(2):143-51. 2. PHENOTYPIC CHARACTERIZATION OF SODALIS PRAECAPTIVUS SP. NOV., A CLOSE NON-INSECT-ASSOCIATED MEMBER OF THE SODALIS-ALLIED LINEAGE OF INSECT ENDOSYMBIONTS Republished with permission of Society for General Microbiology, from [Phenotypic characterization of Sodalis praecaptivus sp. nov., a close non-insect-associated member of the Sodalis-allied lineage of insect endosymbionts, Abhishek Chari, Kelly F. Oakeson, Shinichiro Enomoto, D. Grant Jackson, Mark A. Fisher, Colin Dale, Int J Syst Evol Microbiol, 65, May 2015, 2016; permission conveyed through Copyright Clearance Center, Inc. 4 5 6 7 8 9 10 11 12 13 3. AN INSECT KILLING PHENOTYPE IN SODALIS PRAECAPTIVUS QUORUM SENSING MUTATIONS a a a AUTHORS: Shinichiro Enomoto , Abhishek Chari , Adam.L.Clayton , Colin Dale a a Department of Biology, University of Utah, 257 South 1400 East, Salt Lake City, UT 84112, USA. 14 3.1 Abstract Many bacteria utilize quorum sensing (QS) to mediate control of gene expression in accordance with changes in their cell population density. This affects many important biological processes including the activation of virulence factors that bacteria utilize to effect disease. In the current study, we analyzed the functions of a QS system found in Sodalis praecaptivus, which is a close relative and putative environmental progenitor of the insect-associated Sodalis-allied symbionts, some of which maintain homologous QS systems. Our work shows that mutant strains of S. praecaptivus, lacking critical components of the QS machinery, have a rapid and potent killing phenotype following microinjection into an insect host. Transcriptomic and genetic analyses indicate that insect killing occurs as a consequence of virulence factors, including insecticidal proteins, whose expression is normally suppressed at high cell density by the QS regulatory circuit. Although the mechanistic functions of QS are similar between S. praecaptivus and a derived symbiotic relative, S. glossinidius, genes encoding insecticidal proteins have been lost in the transition to mutualistic associations in the Sodalis-allied symbionts. We discuss the implications of the unusual functionality of this QS system in the context of the origin and evolution of mutualistic relationships involving these bacteria. 3.2 Introduction The regulation of gene expression is an important adaptive trait that allows organisms to modulate their physiology in response to changes in their environment. One such mechanism of regulation in bacteria involves a process known as quorum sensing (QS), in which bacteria synthesize an autoinducer signal (pheromone) that increases in concentration in accordance with bacterial population density (1,2). Using specific transcriptional regulators, bacteria then respond to specific threshold concentrations of the autoinducer in their environment and modulate gene expression accordingly (2). The process of QS is a common method of modulating behavior and physiology in a wide range of bacteria, including pathogens (3). By pairing the expression of many virulence genes with bacterial population density, pathogens can mount an intense attack on their host when their 15 population density reaches a sufficient level to overcome host defenses (4). For example, S. aureus produces exotoxins and tissue-degrading enzymes at the end of its exponential growth phase (5). Similarly, the secretion of exoenzymes contributing to the plant pathogenicity of E. carotovora occurs in a growth phase dependent manner (6,7). However, QS is also known to facilitate mutualistic interactions. For example, in the squid-Vibrio fisheri symbiosis, QS controls the expression of bacterial bioluminescence in the squid light organ, preventing the host from casting a shadow under the moonlit night sky and attracting predators (8,9). Working in concert with a daily cycle of symbiont expulsion and regrowth, this ensures that V. fischeri only achieves a bioluminescent quorum during the hours of darkness (Boettcher, Ruby, and McFall-Ngai 1996; K.-H. Lee and Ruby 1994; Ruby and Asato 1993). In a previous study, we discovered a QS system in the tsetse fly symbiont, Sodalis glossinidius. Microarray analyses showed that the system controlled genes involved in the oxidative stress response, but we were unable to generate a QS-deficient mutant strain of this symbiont to determine the role of its QS system in vivo (13). In the current study, we have focused on a relative of S. glossinidius, known as S. praecaptivus, which was recently discovered in a human hand wound (14). Close relatives of S. praecaptivus have been found in a wide range of insect hosts such as cerambycid beetles (15), archaeococcoid scale insects (16), bird lice (17), spittlebugs (18), potato psyllids (19), mealybugs (20) and louse flies (21,22). In many cases, these bacteria are known to provide beneficial functions for their insect host (23). Comparative genomic analyses indicate that these mutualistic symbionts evolved repeatedly and independently from an S. praecaptivus-like ancestor, undergoing genome degeneration and size reduction as a consequence of the loss of genes that lack adaptive value following the transition to symbiosis (14). In addition, very close relatives of S. praecaptivus have been identified in a range of stinkbugs (24-26), where they seem to exist as transient (i.e. non-maternally transmitted) associates (27), consistent with the notion that their insect hosts serve as vectors for the transmission of these bacteria between plant hosts (14). Since S. praecaptivus is readily amenable to laboratory manipulation, it serves as a 16 platform to investigate the functions of genes that are retained in closely related insect symbionts that are refractory to laboratory culture and/or genetic manipulation. In addition, this approach can yield valuable insight into the nature of the establishment of symbiotic relationships, especially given the fact that the Sodalis-allied symbionts seem to be particularly predisposed to evolving mutualistic associations with insects. In this study, we generated mutant strains of S. praecaptivus that lack the ability to produce or respond to a QS signaling molecule. We then used transcriptomics to determine the identities of genes in the bacterial genome that are regulated by QS. Wild type and mutant bacteria (defective in QS) were then microinjected into a model insect (the maize weevil, Sitophilus zeamais) which is known to have recently developed a symbiotic association with a close relative of S. praecaptivus (28). In contrast to a wild type strain, the QS mutant derivatives demonstrated a potent insect killing phenotype that is associated with the expression of several putative virulence determinants, including two encoding homologs of insecticidal proteins. The QS system was also demonstrated to induce suppression of bacterial growth in vitro, by enhancing expression of a gene (cpmA) with a cryptic amidohydrolase function. Suppression of virulence by the QS system in S. praecaptivus reduces the burden that these bacteria place on their insect host as their population density increases in host tissues, allowing them to maintain a benign and long-lasting infection. 3.3 Results We show that S. praecaptivus and S. glossinidius maintain homologous quorum sensing systems. S. glossinidius has previously been shown, using mass spectrometric analysis, to produce N-(3-oxohexanoyl) homoserine lactone (OHHL) as a QS signaling molecule (13). The synthesis of OHHL is conducted by the protein product of the sogI gene [13], which shares 94% amino acid sequence identity with a gene designated ypeI (locus tag Sant_3558) in S. praecaptivus. Ethyl acetate extracts from both bacteria were separated on a thin layer chromatography (TLC) plate that was overlaid with a culture of an Agrobacterium tumefaciens reporter strain that degrades X-Gal in the presence of exogenous N-acyl HSL, yielding a blue color on the plate (Figure 3.1A). Since the extracts from S. glossinidius and S. praecaptivus each 17 produced a single co-migrating spot in this assay, we conclude that S. praecaptivus also produces only OHHL. We then confirmed that the enzyme encoded by the ypeI gene in S. praecaptivus is required for the synthesis of OHHL by generating a ΔypeI knockout mutant strain using lambda Red recombineering. During growth in a liquid culture, the ΔypeI strain failed to produce any signaling molecule that could be detected using the A. tumefaciens N-acyl HSL reporter strain (Figure 3.1B), confirming that the product of ypeI is responsible for synthesis of OHHL. S. praecaptivus also has two genes encoding candidate LuxR-like response regulators (ypeR and yenR; encoded by locus tags Sant_3587 and Sant_1175, respectively) that share high levels of amino acid sequence identity (93% and 92%, respectively) with previously characterized response regulators found in S. glossinidius (13). The amount of OHHL produced by S. praecaptivus was then measured over 11 hours of growth in liquid LB media, using the A. tumefaciens reporter strain. The amount of OHHL in the culture medium of the S. praecaptivus wild type (WT) strain increased in accordance with population density (Figure 3.1B). The ΔypeI mutant strain growing under the same conditions did not produce any OHHL. However, curiously, it did display an increased growth rate relative to the WT strain. Together these results show that, like S. glossinidius, S. praecaptivus has an OHHL synthase encoded by its ypeI gene, along with a cognate response regulator (encoded by ypeR) and a disassociated second response regulator (encoded by yenR). We conducted a transcriptomic analysis of the genes regulated by OHHL in S. praecaptivus. In order to identify genes whose expression is modulated by quorum sensing, we used a transcriptomic (RNA-Seq) approach to compare gene expression levels in a ΔypeI mutant strain of S. praecaptivus in vitro in the presence or absence of 10 µg/ml OHHL, mimicking a high cell density state (with respect to QS). The 100 most significant results from this RNA-Seq experiment are presented in Table 3.1. A total of thirty genes were found to show significant differential expression with log2 fold changes >2 (Figure 3.2 (A)). The majority of these genes (26/30) demonstrated a decrease in expression in response to OHHL supplementation. Several of these genes are predicted to encode proteins that have functions associated with insecticidal activity and pathogenesis, the most notable of which are those predicted to encode homologs of 18 the PirA and PirB insecticidal toxins (Sant_2443, Sant_2444) that are known to play a role in insect killing in entomopathogenic Photorhabdus spp. (29). Other examples include genes encoding chitinases (Sant_1984, Sant_1963) and chitin binding domain-containing proteins (Sant_1090, Sant_P0292), which may play a role in degrading the insect integument (30) along with collagenase-like proteases (Sant_3444, Sant_3445), which may facilitate infiltration of host connective tissue (31). Several other genes whose transcripts were found to be downregulated in the presence of OHHL (i.e. under quorum) are anticipated to be involved in the formation of respiratory enzyme complexes. These genes encode components of the dimethyl sulfoxide reductase (Sant_0571, Sant_0570, Sant_0569, Sant_0568) and fumarate reductase (Sant_2415, Sant_2416, Sant_2417, Sant_2418) complexes. Other genes whose transcription was downregulated under quorum include a putative deoxyribonucleotide synthase (Sant_3837), two genes whose products are predicted to be involved in nitrogen metabolism (Sant_0171, Sant_2412), two genes whose products are predicted to play a role in regulating cell wall metabolism (Sant_2138, Sant_2857), a tRNA (valY), genes encoding a candidate metallophosphoesterase (Sant_P0288), a putative transcriptional regulator (Sant_3640), a putative colicin receptor (Sant_1851) and a putative citrate-succinate antiporter (Sant_2414). Only four genes demonstrated a significant increase in expression in response to OHHL. Curiously, these include two genes (cpmA and cpmJ; Sant_3163, Sant_3164) that were shown to be induced by OHHL in S. glossinidius and are homologous to components of the carbapenem biosynthesis gene cluster that is present in a range of enteric bacteria (13). Based on the fact that both S. praecaptivus and S. glossinidius only maintain homologs of two genes from this biosynthetic pathway, we concur with the previously established hypothesis that these genes are not involved in antibiotic production and that they have some other unknown function that likely involves the predicted glutamine amidotransferase activity of CpmA (13). The other two genes demonstrating a significant increase in expression in response to OHHL include a putative carbon starvation protein (Sant_2155) and a putative autoinducer 2 kinase (Sant_3797) that may be involved in the regulation of another quorum sensing pathway. In order to confirm the identities of genes regulated by QS, we performed a second 19 transcriptomic comparison using different S. praecaptivus mutant strains lacking each of the QS response regulators (YpeR and YenR). These mutant strains are expected to be impaired in their ability to facilitate the transcriptional changes that occur in the ΔypeI mutant strain when OHHL is added to the culture. Furthermore, by comparing mutant strains that lack each response regulator, we can determine the contribution of each to the process of QS-mediated gene regulation. The data derived from comparative RNAseq analysis of the ΔypeR and ΔyenR mutant strains are presented in Figure 3.2 (A) (for the top 30 most differentially expressed QS-regulated genes) and Table 3.2 (the 100 most significant results). Notably the genes that undergo significant changes in expression between the ΔypeR and ΔyenR strains are a subset of the genes that undergo changes in expression in response to OHHL in the ΔypeI mutant strain. While YpeR and YenR appear to influence the expression of the same genetic targets, the overall directionality of gene expression change indicates that the non-canonical response regulator (YenR) has a more potent impact. This is intriguing because it calls into question the purpose of maintaining two response regulators that modulate the expression of the same target genes. In addition, the amino acid sequences of YpeR and YenR share only 39 % sequence identity, suggesting that they did not arise as a consequence of a recent gene duplication event. In order to validate the RNA-Seq transcriptomic data, we performed chitinase assays in WT S. praecaptivus, along with the ΔypeI, ΔypeR and ΔyenR mutant strains. As expected, the ΔypeI strain had significantly more chitinase activity than a WT strain or a ΔypeI strain grown in the presence of exogenous OHHL (Figure 3.2 (B)). This is consistent with the RNA-Seq data showing that the expression of chitinase genes is significantly reduced in a ΔypeI mutant in the presence of OHHL. The ΔypeR strain has a chitinase activity similar to the WT strain, indicating that YpeR has little influence on the regulation (repression) of chitinase activity under quorum. However, the ΔyenR strain and a ΔyenR ΔypeR double mutant strain had significantly higher levels of chitinase activity under quorum, indicating that YenR is the main response regulator that represses chitinase production under quorum. This fits with the RNA-Seq data showing that the ΔypeR mutant strain had lower numbers of chitinase gene transcripts (particularly for Sant_1984) relative to the ΔyenR mutant, consistent with the role of YenR in repression. 20 We show that the S. praecaptivus QS system controls a weevil killing phenotype. In order to determine how quorum sensing might impact the survival of S. praecaptivus in vivo, we microinjected equal numbers of WT and ΔypeI strains into newly emerged adult grain weevils (S. zeamais) that were previously treated with rifampicin. Rifampicin treatment renders these weevils aposymbiotic by killing their native symbiont, Candidatus Sodalis pierantonius str. SOPE, which is a very close relative of S. praecaptivus (28) and is known to produce OHHL (13). The use of aposymbiotic weevils ensures that the results obtained in the current study only reflect the interaction between the weevil, S. praecaptivus and its endogenous QS system. In addition, symbiont-free weevils are considerably easier to inject because Ca. S. pierantonius str. SOPE provides the weevil with nutrients (aromatic amino acids) that harden the cuticle. Following microinjection, all weevils were maintained under identical conditions on a diet of organic maize in the absence of any antibiotic, and their mobility and survival was monitored daily (Table 3.4). Strikingly, at around one week following injection, weevils that were injected with the ΔypeI mutant strain were observed to become lethargic (Figure 3.3). Over the course of the next seven days, all of the ΔypeI-injected weevils succumbed to death (Figure 3.4 (A)). In contrast, weevils injected with the wild type strain suffered no lethargy or death over the course of experimental monitoring, aside from a small percentage of early deaths resulting from injuries sustained as a consequence of microinjection (<5%). Injection of the ΔypeR and ΔyenR mutant strains (lacking each of the S. praecaptivus QS response regulators) yielded a delayed/reduced weevil killing phenotype in comparison to the ΔypeI strain, with the ΔypeR strain demonstrating slightly more potent killing. However, a ΔyenR ΔypeR double mutant was found to kill as effectively and rapidly as the ΔypeI mutant (Figure 3.4 (A)). Further confirmation of the role of QS in weevil killing was obtained by injecting insects with a 1:1 mixture of WT and mutant bacteria (either ΔypeI, ΔypeR or ΔyenR). In these experiments, WT bacteria largely suppressed the killing effect of the ΔypeI mutant, consistent with the notion that WT bacteria provide a source of exogenous OHHL that effectively complements the ΔypeI mutant strain. However, the co-injection of WT bacteria along with the ΔypeR or ΔyenR mutants did little to mitigate their killing phenoypes, consistent with the notion that the absence of a QS 21 response regulator cannot be complemented in this way (Figure 3.4 (B)). Taken together, the results of these microinjection experiments show that the genes involved in the S. praecaptivus QS system have a significant effect on weevil killing (see Tables 3.3 and 3.4 for data and statistical test results). They indicate that both YpeR and YenR respond to the OHHL synthesized by YpeI and act synergistically to reduce the expression of genes that are responsible for weevil killing, with YpeR having a more potent repressive effect. We identified effector genes that are responsible for weevil killing. In order to identify the QS-regulated genes that are responsible for weevil killing (by the ΔypeI strain), we selected several candidate genes that displayed a significant change in expression in response to OHHL based on the transcriptomic data (Table 3.1) and might be expected to play a role in killing based on their anticipated functions. These candidate genes (from among those listed in Table 3.4) were then knocked out in a ΔypeI mutant background and the resulting double mutants were inoculated into weevils to assess their killing capability. If a candidate gene plays a role in killing, then the resulting double mutant is expected to have a reduced killing capability relative to a ΔypeI single mutant strain. In this experiment, none of the double mutants that were tested in vivo were completely relieved of their weevil killing phenotype. However, the ΔypeI ΔregC, ΔypeI ΔpirA and ΔypeI ΔpirB double mutant strains all demonstrated a statistically significant delay in killing relative to the ΔypeI single mutant (Figure 3.4 (C); Tables 3.3 and 3.4). Furthermore, weevils injected with a ΔypeI ΔpirAB mutant strain were found to have the same median longevity as weevils injected with either ΔypeI ΔpirA or ΔypeI ΔpirB double mutant strains, consistent with the notion that PirA and PirB constitute a binary toxin complex (requiring both toxin components for activity). Furthermore, a ΔypeI ΔpirAB ΔregC triple mutant strain had a less potent killing phenotype than any of the double mutants, suggesting that RegC and PirAB function independently to effect killing. However, even this ΔypeI ΔpirAB ΔregC triple mutant was not completely suppressed in weevil killing, suggesting that there are additional QS-regulated gene(s) that participate in killing that have not yet been identified in our study. Furthermore, although there is a general level of concordance between the transcriptomic data and the identification of genes involved in killing, it should be noted that our in vitro transcriptomic analysis revealed that 22 YenR has a more potent repressive effect on the transcription of pirA, pirB and regC under quorum (Table 3.2). However, the ΔypeR strain has a more potent killing phenotype in vivo. This raises the possibility that the functions of the response regulators are different in vivo, at least with respect to their relative contributions to the process of QS-mediated gene regulation. While it is beyond the scope of the current study to examine the role (in killing) of all genes that are differentially regulated by QS in S. praecaptivus, we did elect to test the effect of a ΔypeI ΔSant_1962 double mutant, because Sant_1962 shares a high level of sequence identity with genes that are known to encode insecticidal delta endotoxins. In addition, its expression is reduced under quorum like that of pirA and pirB, albeit to a lesser extent (adjusted p = 0.09; Table 3.1). However, weevils injected with the ΔypeI ΔSant_1962 double mutant had the same median lifespan as weevils injected with the ΔypeI single mutant (Table 3.4), suggesting that Sant_1962 is not an effector of the QS-regulated mechanism of killing documented in this study. All other genes in the S. praecaptivus genome that are predicted (by virtue of homology) to function as toxins (Sant_0919, Sant_1053, Sant_2840) did not display significant reduction in expression in the presence of OHHL in the in vitro transcriptomic analysis (Table 3.1). We show that quorum sensing suppresses the growth of S. praecaptivus in vitro. During the course of our experiments, we noticed that the rate of growth of the S. praecaptivus WT strain in liquid LB media was consistently lower than that of the ΔypeI mutant (Figure 3.1 (B)). Furthermore, the addition of exogenous OHHL to LB plates was found to have a significant inhibitory effect upon the growth of both the S. praecaptivus WT and ΔypeR mutant strains (Figure 3.5 (A)). However, the growth of the S. praecaptivus ΔyenR mutant was not suppressed by exogenous OHHL (Figure 3.5 (A)). In addition, the introduction of a plasmid overexpressing the OHHL synthase (YpeI) could only be achieved in a ΔyenR strain, presumably because the overexpression of this gene in a WT strain induces a severe growth defect, due to overproduction of OHHL. Furthermore, when this YpeI-overexpressing (ΔyenR) strain was streaked onto LB agar, it inhibited the growth of adjacent WT and ΔypeR (but not ΔyenR) colonies (Figure 3.5 (B)). Since the ΔyenR mutant still produces OHHL due to the fact that it maintains an intact ypeI gene, the increased growth rate of the ΔypeI mutant (relative to WT) cannot simply be explained as a 23 consequence of the loss of the metabolic burden associated with synthesizing OHHL. Rather, these results suggest that OHHL interacts with YenR, to modulate the transcription of target gene(s) that result in a suppression of growth. Since none of the mutants that were screened in the weevil killing assays demonstrated an increased growth rate relative to the WT strain, we turned our attention towards genes that demonstrated a significant increase in transcript levels in the presence of OHHL. On LB agar plates supplemented with OHHL, we found that ΔcpmA and ΔcpmAJ mutant strains had an elevated growth rate relative to the WT strain (Figure 3.6). However, since a mutant lacking cpmJ alone demonstrated the same growth suppression as the WT strain in the presence of OHHL, it is clear that cmpA alone is responsible for the growth suppression phenotype. Furthermore, the ΔcpmA strain grew just as well as the ΔyenR strain in the presence of OHHL, in both solid (Figure 3.6) and liquid media (Figure 3.7), suggesting that cpmA is the only gene mediating the QSassociated growth suppression phenotype observed in our study. We show that quorum sensing modulates bacterial population density in vivo, but not via CpmA. Given that the quorum sensing system in S. praecaptivus induces growth suppression in vitro, we sought to determine if it also affects the density of bacterial infection in vivo, following microinjection into weevils. This is an important question because an elevated density of bacterial infection would be anticipated to yield increased quantities of the PirAB toxin that is known to be involved in weevil killing. To investigate this issue, bacterial infection densities were estimated (only from live weevils) by plating the homogenates of surface sterilized insects on selective media and counting the colony-forming units obtained at intervals following microinjection. The resulting data (Figure 3.8 (A)) show that the WT strain maintains a consistent infection density in the weevil over the course of its lifespan, which equates to the lifespan of uninfected weevils (Table 3.4). However, the ΔypeR strain displays a >10-fold increase in infection density (relative to WT) over the course of four weeks following injection and the ΔyenR strain achieves a ~2-fold increase (relative to WT) over the same time period. It should be noted that we could not obtain sufficient data for the ΔypeI strain beyond two weeks following microinjection due to its potent killing phenotype. Contrary to what was observed in the in vitro experiments, the ΔcpmAJ strain 24 did not display an elevated growth in vivo, maintaining a similar infection density to the WT strain. In addition, the deletion of cpmAJ had no effect on weevil killing either when the mutation was introduced into a WT background or into a ΔypeI background (Figure 3.4 (C)). Rather, the significant increases in infection densities observed with the ΔypeR and ΔyenR strains are more readily explained as a consequence of the increased virulence properties of these mutants, highlighted by the fact that the increases in infection densities are most notable in the latter stages of infection, when the weevils are sick, presumably as a consequence of prolonged exposure to bacterial toxin(s) (Figure 3.8 (B)). These results, however, do not rule out the possibility that cpmA has a beneficial role in symbiosis. To this end, it is notable that the cpmA gene has been retained in an intact form in two insect symbionts that are closely related to S. praecaptivus (Figure 3.2 (A)). 3.4 Discussion In the current study, we show that S. praecaptivus maintains an N-acyl HSL-based QS system that is genetically homologous to a system characterized previously in the derived insect symbionts, S. glossinidius and Ca. S. pierantonius str. SOPE (13). Microarray analysis of the S. glossinidius transcriptome revealed that the QS signaling molecule (OHHL) induced a substantial increase in the transcription of two genes of unknown function (cpmA and cpmJ) along with minor increases in the transcription of a wide range of genes involved in the cellular oxidative stress response (13). However, the inability to generate a mutant strain of S. glossinidius, lacking the OHHL synthase gene (sogI), compounded the analysis of the functions of this QS system in vivo. In the current study, we used an array of genetic and transcriptomic tools to investigate the homologous QS system in S. praecaptivus, yielding surprising insight into its functions in vivo. Transcriptomic analysis of QS-defective mutant strains revealed that, at high bacterial population density, the S. praecaptivus QS system has a generally repressive effect upon the transcription of genes under its control. Furthermore, these analyses revealed that the QS system of S. praecaptivus does not significantly increase the transcription of genes involved in the bacterial oxidative stress response, indicating that this is a derived characteristic in S. glossinidius. We did however find that the QS system of S. praecaptivus drives an increase in 25 transcription of the enigmatic genes, cpmA and cpmJ, as previously observed in S. glossinidius (13), indicating that there is some functional synergy in the QS systems that are shared by these bacteria. Furthermore, in the current study, we found that cpmA mediates suppression of bacterial growth in vitro in response to the QS signal, OHHL. This suppression is eliminated in mutant strains lacking either cpmA or the gene encoding the QS response regulator (YenR) that increases cmpA transcription in response to OHHL. However, no enhancement of bacterial growth was observed following injection of a cpmA mutant strain into weevils. Curiously, in other Gram-negative bacteria, with the exception of members of the Sodalis-allied clade, homologs of cmpA and cmpJ constitute components of larger gene clusters encoding enzymes involved in the biosynthesis of the antibiotic, carbapenem (32,33). In this context, CpmA (also known as CarA in some bacteria) catalyzes the formation of a β-lactam ring using domains homologous to those of β-lactam synthase (B-LS) and asparagine synthetase (ASN) (34). The S. praecaptivus and S. glossinidius homologs of CpmA are highly conserved and both lack the region of the polypeptide encoding the B-LS domain, suggesting that they are not involved in β-lactam synthesis. Conservation of the ASN domain suggests that the function of CpmA involves amidohydrolase activity, which is known to be involved in a wide range of physiochemical processes in nature (35). The transcriptomic analyses presented in this study show that the QS system of S. praecaptivus has some unique properties that have likely been lost in S. glossinidius as a consequence of the genome degeneration process that accompanies the transition to a static and obligate insect-associated lifestyle (14). Principal among these is the propensity for QS to downregulate the transcription of genes that are anticipated to have virulence properties, including insecticidal toxins and enzymes that are anticipated to degrade the insect integument. It was therefore of great interest to compare the effects of injecting WT and QS-deficient mutant strains of S. praecaptivus into an insect host. To achieve this goal, bacteria were injected into aposymbiotic maize weevils. This led to the striking finding that QS-deficient mutants have a potent weevil killing phenotype that is due, at least in part, to the action of genes encoding homologs of the binary insecticidal Pir toxins. 26 Genes encoding Pir toxins were initially identified in the entomopathogen Photorhabdus luminescens (29) and subsequently found in other bacteria including Vibrio parahaemolyticus, in which they play an important role in shrimp pathogenesis (36). It was shown that the crystal structure of the V. parahaemolyticus Pir toxins resembles that of the Bacillus thuringiensis Cry toxins, suggesting that they may have a conserved mode of action that ultimately causes destabilization of the host cell cytoskeleton, leading to cell death (37). Interestingly, S. praecaptivus also possesses a gene encoding a putative protein that shares substantial sequence identity with a Cry toxin. However, transcription of that gene is not controlled by QS and does not appear to play a role in the weevil killing phenotype described in this study. The only other gene identified to affect weevil killing in a QS-defective strain, annotated as regC, shares substantial sequence and structural homology with the bacteriophage P2 repressor that has a regulatory function. Thus, it seems unlikely that RegC is a direct effector of killing and more likely that it serves as a secondary regulator in the QS circuit, modulating the transcription of other genes that effect killing, perhaps including pirA and pirB. Indeed, a mutant strain lacking ypeI, pirA, pirB and regC displays a delayed killing phenotype relative to either a ΔypeI ΔpirAB or a ΔypeI ΔregC mutant strain, implying that RegC does influence the expression of effectors of killing other than just PirA and PirB. Clearly, further transcriptomic analyses are required to define the role of RegC in the process of killing. Moreover, based on the fact that the inactivation of pirAB and regC only delays the killing of weevils, rather than completely eliminating killing, it seems likely that there are additional virulence loci that remain to be identified that play a role in this process. Alternatively, it is possible that the weevil killing phenotype relies on a complex interplay of effectors that could not be investigated using our simple genetic screen. It is pertinent to note that QS-mediated gene regulation is often used by bacterial pathogens to activate the expression of virulence factors at high bacterial population densities, in order to induce a concerted and potent attack on the host (4,38). However, our work shows that S. praecaptivus utilizes QS to reduce virulence gene expression when bacterial infection density increases in the host insect. This decoupling of virulence and infection density clearly facilitates S. praecaptivus maintaining a high density of infection in its host whilst minimizing negative 27 impacts on host fitness or longevity. Such a trait is anticipated to be particularly important for vector-borne parasites that need to maximize the fitness of their hosts as agents of dispersal (39). Indeed, it has been suggested that vector-borne parasites might even evolve positive impacts on host fitness to offset the metabolic cost of their passage (40). In such a scenario, a switch to vertical transmission would then be anticipated to effect the establishment of a mutualistic relationship. While the ecological underpinnings of the associations between Sodalis-allied symbionts and their insect hosts are not fully understood, it is clear that these bacteria have repeatedly and independently evolved mutualistic associations with insects that feed on a diverse array of plant and animal hosts (14). Based on the fact that S. praecaptivus was isolated from a human host and that its genome maintains a number of virulence genes that are normally found in plant pathogenic bacteria, we postulated that such mutualistic associations might have arisen from a context of vectorial transmission. Furthermore, close relatives of S. praecaptivus that have been found in certain stinkbugs and chestnut weevils have a low prevalence of infection within insect populations, consistent with the notion that these bacteria are not maternally inherited (in contrast to their mutualistic relatives) but are instead acquired from the environment by each new insect generation, presumably as a consequence of feeding on infected trees (24,27,41). Based on the results of our study, it is also important to acknowledge that genes such as pirAB and regC must have a beneficial function in S. praecaptivus when they are expressed at low bacterial population densities. Given that low bacterial population density likely exists when insect hosts are initially infected, one explanation is that such virulence genes are required to assist bacteria in gaining entry into host tissues. Alternatively, these genes might play a role in host defense. In an elegant series of studies focusing on aphids, it was shown that a symbiotic bacterium named Hamiltonella defensa utilizes toxins to kill parasitoid wasps that attack its aphid host (42). The toxins utilized in this process are encoded by lysogenic bacteriophages (viruses) that are maintained by H. defensa and apparently induce relatively frequent bacterial cell lysis in order to synthesize (and liberate) their parasitoid-killing, toxic cargo (43). While we cannot rule out the possibility that S. praecaptivus or a derived, allied symbiont plays an analogous role in host defense, there are no such examples that have been reported to date involving these 28 bacteria. In addition, even the recently derived symbionts that are closely related to S. praecaptivus have lost many of the putative virulence genes (including pirAB) that are highlighted in our study, presumably as a consequence of the transition to permanent insect association, which removes the requirement for symbionts to participate in the initiation of infection. 3.5 Materials and Methods Weevil rearing and maintenance were done as follows. S. zeamais weevils were reared on organic whole yellow maize (Purcell Mountain Farms) in an incubator at 27°C and 60% relative humidity. Symbiont-free (aposymbiotic) weevils were generated by rearing weevils on rifampicin treated corn that was prepared by hydrating dried corn with a 3% (w/v) solution of rifampicin (1mg/ml), prior to addition of insects. After three generations on this antibiotic diet, the loss of symbionts was confirmed by microscopic examination of 100 larvae (revealing absence of bacteriomes) and by negative PCR assays on whole weevil DNA using symbiont specific primers. Aposymbiotic insects were subsequently maintained on a normal diet and checked periodically to confirm the absence of symbionts. Bacterial culture and genetic modification were done as follows. All strains of S. praecaptivus were cultured in LB media with appropriate antibiotics, unless otherwise described. Gene disruptions/deletions in S. praecaptivus were achieved using the lambda Red recombineering procedure, as described previously (44). Approximately 150 nucleotides of the 5' and the 3' of target genes were amplified by PCR with Phusion High Fidelity DNA Polymerase (Fisher Scientific). The 5' and 3' flanking sequences were then joined to an antibiotic resistance cassette (encoding either kanamycin, spectinomycin or gentamycin resistance) by PCR with Taq Polymerase MasterMix based on a previously described protocol [44]. The resulting construct was then electroporated into an S. praecaptivus strain harboring the lambda Red-Gamm plasmid, pRed/Gamm (CAT). For transformation, S. praecaptivus cultures were grown in 25 ml 2YT5.8 media (20g/l Tryptone, 8g/l Yeast Extract, 10g/l NaCl, adjusted to pH 5.8) until their OD600 reached 0.4 units. Expression of lambda Red-Gamm proteins was then induced by addition of 0.4% arabinose for 30 minutes. Cells were harvested and washed twice in sterile ice cold water. DNA was 29 electroporated into cells using an Eppendorf electroporator model 2510, at 1600 V/s. Following a 1-hour recovery in SOC media, recombinant clones were then selected by plating on LB media with the appropriate antibiotic(s). Gene disruptions were verified by testing the linkage between the antibiotic resistance marker and the bacterial chromosome using PCR. Cultures of the Agrobacterium tumefaciens KYC55 (pJZ372) (pJZ384) (pJZ410) reporter strain were grown in AT minimal media at 28°C (46), supplemented with 1.5µg/ml of tetracycline, 100 µg/ml of gentamycin and 100 µg/ml of spectinomycin (13). OHHL extraction and TLC assay were performed as follows. OHHL extraction was performed on culture supernatants based on a previously described procedure [45]. Briefly, 25 ml cultures of S. glossinidius (grown in liquid MM medium, at 25°C and 200 rpm shaking) and S. praecaptivus (grown in LB medium, at 30°C and 200 rpm shaking) were pelleted by centrifugation (8,000g, 20 min., 4°C) once their turbidity reached an O.D600 of 0.4 units. The resulting culture supernatants were filtered through 0.22 micron pore-size membrane filters (Argos Technologies) and then extracted by shaking with an equal volume of ethyl acetate for 30 minutes. The ethyl acetate extracts were dried with anhydrous magnesium sulfate and filtered. The solvent was then evaporated using a vacuum centrifuge at 60°C and the OHHL-containing residue was resuspended in 100 µl of methanol. These extracts were then subjected to thin layer chromatography on a C18 reverse-phase TLC plate (Whatman) using methanol:water (60:40) as the solvent/carrier. Following development, the plate was dried and overlaid with a live culture of an A. tumefaciens acyl-HSL reporter strain as described previously [46]. OHHL bioassays were done as follows. The wild type and ΔypeI strains of S. praecaptivus were cultured overnight in 10 ml LB liquid media from single colony inoculations. These cultures were washed, resuspended and used to start 50 ml cultures, at OD 600 of 0.03 units. During an 11-hour incubation with shaking at 200 rpm and 30°C, the cultures were sampled every 2 hours to check cell density and assay for the presence of OHHL with the β-galactosidase activity (Miller) assay (48) using an A. tumefaciens acyl-HSL reporter strain, KYC55 (pJZ372)(pJZ384)(pJZ410), as described previously [46]. The extraction of OHHL was performed from 900 µl of supernatant from the S. praecaptivus cultures into 100 µl of methanol. Following 30 this, 5ml cultures of the A. tumefaciens acyl-HSL reporter strain (starting OD 600 of 0.03) were exposed to 10 µl of the OHHL extracts for 12 hours at 200 rpm shaking, prior to performing βgalactosidase assays. OHHL concentration was also measured for S. praecaptivus wild type cultures of 20 ml, grown for four hours at 200 rpm shaking and 30°C to OD 600 of 0.4. The extraction of OHHL was performed from total culture supernatant into 200 µl of methanol, which was then used as substrate in the A. tumefaciens /β-galactosidase assay. Solutions of 50, 500 and 1250 nM OHHL (Sigma Aldrich, K3255) were prepared in methanol for use as standards. The concentration of OHHL in the OD 600 = 0.4 culture was determined to be ~1 µg OHHL/ml. Transcriptomics assays were performed as follows. 20 ml cultures of S. praecaptivus were grown in LB media, at 200 rpm shaking and 30°C, with or without supplementation with OHHL (10 µg /ml), which represents ten times the concentration found in the S. praecaptivus culture at an O.D 600 = 0.4, based on the bioassay described above. Total nucleic acid extraction was performed on each culture as described previously, after four hours of growth for the experiment involving the ΔypeI strain or five hours of growth for the experiments involving the ΔypeR and ΔyenR strains (49). RNA was extracted using the Purelink RNA minikit (Ambion) according to the manufacturer's instructions. The resulting RNA was analyzed using an Agilent bioanalyzer to ensure that it lacked DNA contamination and was of sufficient quality for cDNA preparation. Total RNA samples were then submitted to the core sequencing facility at University of Utah for Illumina TruSeq library preparation and sequencing on a HiSeq instrument (Illumina). The resulting 50-base single reads were filtered using NGSQCToolkit (50) and mapped to the reference genome sequence using bowtie2 (51) employing the --very-sensitive preset. Read counts mapping to individual genes were generating using HTSeq (52) by running htseq-count using the options -s no -a 0 -m intersection-nonempty -t gene. Next, using custom Perl scripts, counts from three biological replicate cultures were combined and subject to statistical analysis. After normalization, combined counts files were compared using DESeq2 (53). Chitinase assays were done as follows. Chitinase activities in the S. praecaptivus culture supernatants were measured using a fluorimetric assay (Sigma-Aldrich, CS1030) according to the manufacturer's instructions. Cells were grown in LB media (15 ml volume), from a starting OD 31 600 of 0.05, with 200 rpm shaking at 30°C until O.D 600 reached ~1 unit. Then, 10 µl of culture supernatant was added to 90 µl of the substrate working solution (containing 4-methylumbelliferyl β-D-N, N-diacetylchitobioside hydrate) in a 96-well fluorescence plate (Greiner Bio-One, 655096) and incubated for 60 minutes at 37 °C in the dark. The reaction was stopped by adding 200 µl of sodium carbonate buffer, and the fluorescence was measured with a SynergyMx plate reader (BioTek Instruments Inc.) within 15 minutes of the reaction end point. The chitinase activity was calculated using the 100 ng 4-methyl umbelliferone standard. Growth suppression assays were performed as follows. Plate growth assays were performed by spotting appropriate dilutions of overnight cultures, on plates containing LB agar without sodium chloride (to inhibit swarming motility). OHHL supplementation was achieved by pipetting OHHL directly onto plates or impregnating sterile paper strips on the surface of the plates after soaking them in 10 mg/ml OHHL in methanol. Controls were performed using an equivalent volume of methanol alone. Recombinant S. praecaptivus strains were also used as a source of OHHL by streaking bacteria onto plates for testing. One of these recombinant strains maintains a pCM66 plasmid expressing ypeI in a ΔyenR background and can therefore overproduce OHHL without undergoing YenR-mediated growth suppression. The other strain maintains an empty pCM66 plasmid in a ΔypeI background and therefore does not produce any OHHL. All plates in these experiments were incubated at 30°C. Liquid culture assays were done by growing the appropriate bacterial strains until their OD600 reached 0.3 and then splitting them in two equal volumes of 20 ml and supplementing the resulting cultures either with 200 µl of 10 mg/ml OHHL (100 µg/ml final concentration in media) or an equal volume of water. The turbidity of each culture was then monitored over a five hour period during incubation with shaking at 200 rpm at 30°C. Weevil microinjections were performed as follows. Newly emerged adult aposymbiotic weevils were used for all microinjection experiments because these insects have a significantly thinner cuticle relative to their symbiotic counterparts, facilitating a substantially higher level of survival following microinjection. Capillary tubes (3.5" Drummond # 3-000-203-G/X, Drummond Scientific Company) were pulled to create a short sharp tip (Bee-Stinger needle) with Micropipette 32 puller P-97 (Sutter Instruments). The settings on the micropipette puller were as follows: Heat=292, Pull=100, Velocity=60, Time=250, Pressure=500. Weevils were immobilized by a brief incubation on ice and the needle was immersed in 50µl of an overnight bacterial culture to collect bacteria by capillary action. The needle was then used to pierce the cuticle of the weevils in the thoracic region between the middle and hind legs. This resulted in a median 2800 bacterial cells being transferred into each weevil, as observed by plating weevil homogenates directly after injection. Following injection, weevils were then transferred to hydrated maize grains and maintained in an incubator at 27°C and 60% relative humidity. The cultures were inspected weekly to assess weevil mortality following injection. Bacterial isolation from weevils was done as follows. Surface contaminants were removed from weevils by immersing the adult insects in a solution of 10% bleach for 5 minutes with agitation using a magnetic stir bar. Weevils were then air dried and homogenized in 100µl of sterile water. A dilution series was plated on LB agar (without sodium chloride) containing 50µg/ml polymyxin B to select for S. praecaptivus. Weevil morbidity assessment was performed as follows. Individual weevils were placed in the center of an empty petri dish and the time taken for them to move from the center of the dish to the periphery was recorded. Any weevils that displayed no sign of movement during the assay were excluded from the experiment. 3.6 Acknowledgements We thank Kelly Hughes for the provision of plasmids used in this study. 33 Figure 3.1. Characterization of the S. praecaptivus QS system. (A) TLC plate with ethyl acetate extracts derived from culture supernatants of S. praecaptivus and S. glossinidius. The plate was overlaid with an A. tumefaciens AHL reporter strain that produces blue spots in the presence of AHL molecules. This assay shows that S. praecaptivus and S. glossinidius each produce a single AHL molecule, which was previously characterized as OHHL in S. glossinidius (13). (B) Growth (OD600) and OHHL production (assayed using the A. tumefaciens reporter strain) of WT S. praecaptivus and an isogenic strain lacking the putative OHHL synthase (ΔypeI). Data were obtained from three biological replicates and error bars show standard deviations. - dmsA1 frdA pirA - pirB frdB Sant_P0292 Sant_1984 Sant_0571 Sant_2415 Sant_2444 Sant_2414 Sant_2443 Sant_2416 Fumarate reductase subunit B JHE-like toxin 530.119 171.035 554.55 20.78 51.98 681.4384 583.5851 689.852 42.394 87.066 1452.065 833.559 2616.483 66.5 153.99 Putative anion transporter 1999.05 58.19 129.9 97.46 171.3617 142.8421 192.4725 7.4814 20.142 1558.289 591.103 66.5 JHE-like toxin Fumarate reductase subunit A 2027.63 3105.555 2541.09 3359.693 177.9 241.06 Put. Dimeth. sulfox. Reduct. 1472.107 758.379 major subunit Putative exochitinase 3353.98 112.2 155.3 11.35 43.5 126.7 5.674 68.09 105 143.7 53.91 ct ct ct ct ct ct OHHL NOOHHL NOOHHL NOOHHL OHHL OHHL 1 3 2 1 3 2 Fibronectin type III domain3161.674 2386.97 cntnng. protein Predicted product log2 Fold change 1.23E-11 2.92189 8.87E-20 3.02496 9.67E-16 3.09688 6.73E-17 3.16055 2.40E-16 3.21556 2.19E-21 3.36117 5.36E-33 3.56756 1.70E-32 4.06066 padj Note: Cultured in the presence or absence of OHHL, ranked according to log2fold change in transcript numbers (first hundred results). Three biological replicates were performed for each condition and the columns containing count data are labeled ctOHHL1-3 (for cultures with OHHL) or ctNOOHHL1-3 (for cultures lacking OHHL). Gene name Locus Table 3.1. OHHL dependent transcriptomes of ΔypeI mutant strain. 34 frdD Sant_2418 frdC ct ct ct ct ct ct NOOHHL NOOHHL OHHL OHHL NOOHHL 1 OHHL 1 3 2 3 2 padj log2 Fold change 79.8788 272.51059 8.3126 29.239 4.7286 1.81E-09 2.70796815 3157.665 2090.005 4975.7003 231.09 435.98 347.08 1.14E-15 2.83060008 Fumarate reductase subunit C 219.4633 Outer membrane protein Fumarate reductase subunit D 209.4421 62.96329 222.96321 9.1439 18.843 10.403 1.19E-11 2.83325959 Predicted product - - Sant_0568 dmsD Sant_1963 Sant_0569 dmsC Sant_1090 Twin-argninine leader-binding protein Chitinase 19.86 1.29E-10 2.49229026 166.3511 58.26454 165.79315 13.3 19.492 17.969 4.12E-09 2.3640079 99.20942 54.50553 108.6231 14.131 11.695 8.5114 6.19E-10 2.39133524 221.4675 84.57755 219.15187 17.456 25.99 DMSO reductase anchor subunit 74.2403 85.755079 9.9751 13.645 5.6743 6.54E-12 2.58890469 102.2158 Putative chitinbinding domain 3 protein Sant_3640 regC Prophage P2 protein 148.3131 98.67381 163.88749 17.456 17.543 9.4571 1.03E-13 2.68123125 Sant_2417 Sant_1851 ompW Gene name Locus Table 3.1. (Continued) 35 nasB - lytS Sant_2412 Sant_P0288 Sant_3445 Sant_2138 yhbU - - Sant_3444 Sant_3837 Sant_0171 Sant_0570 dmsB Gene name Locus padj log2 Fold change 325.68749 219.9016 617.43657 49.876 55.878 64.31 4.86E-10 2.322843 ct ct ct ct ct ct NOOHHL NOOHHL NOOHHL OHHL OHHL OHHL 3 3 2 1 2 1 117.24749 76.1198 123.86845 19.119 15.594 16.08 4.60E-10 2.2445433 204.43153 107.1316 230.58588 21.613 29.239 32.15 4.27E-10 2.2861539 282.59653 179.4924 491.66246 35.744 70.173 59.58 9.85E-08 2.0846682 Anaer. ribonuc.triphosph. reductase Nitrate reductase 2 alpha 937.97996 510.2846 1248.2128 147.96 217.67 127.7 1.61E-08 2.0804602 subunit 199.42095 140.9626 259.17091 27.432 30.538 48.23 2.23E-09 2.1386482 Peptidase U32 Anaer. Dimeth. Sulfox. 325.68749 138.1433 343.02032 35.744 62.376 26.48 3.71E-08 2.1938857 reductase chain B Signal transduction histidine kinase Peptidase U32 Metallophosphoesterase 317.67056 217.0824 352.54866 42.394 64.975 32.15 6.50E-11 2.2867733 Nitrite reductase Predicted product Table 3.1. (Continued) 36 Gene name valY fruB - - ycfP ynfK uxaC Locus valY Sant_1414 Sant_2857 Sant_ps0811 Sant_0172 Sant_2464 Sant_2000 Sant_3652 9.975 11.695 6.62 log2 Fold change 1.975177 595.2565 577.94661 1131.967 100.6 259.25 102.14 Glucuronate isomerase NA 1.893857 52.63 75.657 6.47E-08 1.898075 191.404 265.94942 365.8883 52.37 Putative dethiobiotin synthase 46.34 6.60E-08 1.973141 NA 153.3236 137.20359 299.1899 29.09 42.234 46.34 2.38E-07 1.953524 77.32 2.067981 4.30E-07 2.031905 NA Hypothetical protein Cryptic nitrate reductase 2 331.7002 182.31161 392.5677 58.19 subunit beta 32.06769 20.674513 47.64171 4.156 4.5482 7.5657 Putative phage 56.11846 40.409275 66.6984 lysozyme - padj 185.3913 268.76867 409.7187 59.85 34.437 64.308 3.02E-08 2.068508 ct ct ct ct ct ct NOOHHL NOOHHL NOOHHL OHHL OHHL OHHL 1 3 2 1 3 2 PTS fam. fruct. porter; IIA/HPr 65.1375 59.204287 202.0009 16.63 14.944 19.86 component - Predicted product Table 3.1. (Continued) 37 Gene name valU - - valX csrB - Locus Sant_0173 valU Sant_2137 Sant_3838 valX Sant_2623 csrB Sant_2803 ct ct ct ct NOOHHL NOOHHL NOOHHL OHHL 3 2 1 3 ct OHHL 2 ct OHHL 1 padj log2 Fold change 3816.055 5434.578 7891.373 1141.3 1402.8 1517.9 7.51E-08 1.825254 128.2708 96.79431 114.3401 17.456 37.036 23.643 1.20E-06 1.79851 Putative chitinase 95.20096 90.21606 156.2648 24.107 32.487 17.023 1.07E-06 1.848795 hypothetical protein - 237.5013 356.1655 466.8888 84.789 64.975 91.734 8.16E-08 1.860095 11.638 11.046 12.294 2.22E-06 1.861793 - 72.4154 142.3004 53.56578 125.7741 22.444 22.091 22.697 3.07E-06 1.861865 AraC family transcriptional regulator Anaer. ribonuc.triphosph. red. 49.10365 46.04778 Act. protein 129.2729 172.9141 301.0956 54.863 24.041 47.286 3.04E-06 1.865321 - Nirate reductase 123.2602 64.84279 150.5478 21.613 31.188 16.077 2.22E-06 1.88066 delta subunit Predicted product Table 3.1. (Continued) 38 - 49.10365 27.25277 53.358716 9.14388 14.944 6.62 NA 1.618989 hypothetical protein Sant_0455 - 1936.087 1612.612 2498.3313 541.983 398.95 679.97 4.30E-07 1.683185 142.8421 259.17091 41.5631 46.782 52.014 1.15E-06 1.723207 Cytochrome d ubiquinol oxidase subunit I 156.33 Sant_2767 cydA1 2-isopropylmalate synthase 1554.281 2022.343 3904.7146 527.851 831.68 449.21 1.17E-05 1.721179 ssrS 1.7629 1;4-dihyd.-2-naphth. 194.4104 205.8054 384.94502 59.0196 59.777 71.874 1.03E-06 1.759215 octaprenyltransferase 250.5288 268.7687 442.11508 69.826 59.777 104.03 1.03E-06 - ssrS Sant_3366 leuA Sant_3971 Peptidase T Sant_2456 pepT log2 Fold change 1.89E-06 1.796928 padj 327.973 426.86973 70.6573 128.65 70.928 3.48E-07 1.768271 348.7361 66.2 ct ct OHHL OHHL 2 1 D-galactonate transporter ct OHHL 3 Sant_3653 exuT ct ct ct NOOHHL NOOHHL NOOHHL 3 2 1 319.6748 314.8164 512.62481 63.1759 133.85 Predicted product Altronate oxidoreductase Gene name Sant_3651 uxaB Locus Table 3.1. (Continued) 39 150.7 95.517 NA 1.562429 log2 Fold change YfbU family protein 1-phosphofructokinase Sant_1336 yfbU Sant_1415 fruK 157.8781 304.90695 49.876 61.08 69.983 4.00E-05 1.540774 41.08673 16.915511 142.92513 20.782 12.99 11.349 0.0064197 1.49739 165.349 Cytochrome d ubiquinol 1091.304 863.63079 1358.7416 367.42 229.4 389.63 7.43E-06 1.542728 oxidase subunit II Sant_2766 cydB1 2818.95 1615.4313 4413.5281 748.97 501.6 1169.8 0.0002668 1.547153 L-1;2-prop. oxidoreduct.; 207.4379 140.96259 371.60534 52.369 92.91 51.068 0.0002452 1.543881 lactald. reductase Formate acetyltransferase 1 Sant_3781 fucO Sant_2637 pflB DNA starv./station. phase 1072.263 805.36626 1827.536 169.58 544.5 262.91 0.0004569 1.556644 protect. protein 281.5944 286.62393 623.15358 78.97 padj Sant_2688 dps hypothetical protein ct ct ct ct ct ct NOOHHL NOOHHL NOOHHL OHHL OHHL OHHL 3 2 1 3 2 1 Bifunct. acetald.-CoA/alc. 695.468 608.01863 2111.4806 233.58 366.5 293.17 0.000508 1.557598 dehydrogenase - Sant_0800 Predicted product Sant_1834 adhE Gene name Locus Table 3.1. (Continued) 40 - Sant_2569 117.2475 46.98753 137.2081 19.95 38.335 28.371 0.00139881 1.4456 Formate transporter 316.6684 260.3109 398.2847 97.258 63.675 150.37 0.00030847 1.41556 Sant_2636 focA 161.3406 129.6856 242.0199 45.719 85.767 27.426 0.00139578 1.42099 Tag.-6-phosph. ket./ald. isomerase 2-s.-5-e.-6-h.-3-c.-1113.239 117.4688 188.6612 35.744 40.284 55.797 0.00016187 1.43738 carboxylate synthase Nitrate reductase gamma subunit 39.0825 37.59002 78.13241 14.131 5.8477 22.697 0.00308128 1.44909 0.00199501 1.45574 Sant_0025 agaS1 Sant_1357 menH Sant_0174 narV hypothetical protein TIM-barrel signal 145.3067 187.0104 306.8126 37.407 107.86 transduction protein - Sant_2184 33.1 99.20942 82.69805 123.8684 39.069 18.193 34.991 0.00024053 1.47094 cyd operon protein YbgT 1.4842 Sant_2765 ybgT 2.39E-05 112.2369 101.4931 146.7365 36.576 37.685 36.883 Acetolactate synth. isozyme II large subunit log2 Fold change Sant_0277 ilvG padj ct ct ct ct ct ct NOOHHL NOOHHL NOOHHL OHHL OHHL OHHL 3 2 1 3 2 1 Predicted product Locus Gene name Table 3.1. (Continued) 41 ct ct OHHL OHHL 2 1 5.0105767 6.578254 5.7170053 0 0.6497 1.8914 - Sant_0024 agaV Sant_2183 7.35E-05 NA 7.35E-05 NA 1.356026 1.357202 1.364823 1.372173 59.777 14.186 0.00773629 1.336626 145.30672 187.0104 312.52962 39.901 118.25 39.72 0.00495398 1.351306 NAG-spec. enz. 79.167112 90.21606 142.92513 19.95 IIB cmpnt of PTS hypothetical protein Ribonuclease Sant_3972 rraA activity regulator 353.74671 404.0928 596.47422 152.12 155.94 162.66 protein expressed protein ilvX NAD(P) Sant_1922 pntA transhydrogenas 681.43843 604.2596 1027.1553 230.26 284.59 282.77 e subunit alpha Sant_0276 ilvX log2 Fold change 266.4 127.67 0.00161072 1.378018 Put. phage-rel. transmem. 23.048653 14.09626 19.056684 5.8188 7.1472 1.8914 protein - padj 143.30249 128.7458 243.92556 58.188 53.929 57.688 0.00024053 1.38511 ct OHHL 3 Sant_0812 Putative phosphatase ct ct ct NOOHHL NOOHHL NOOHHL 3 2 1 Bacterioferritin 393.83133 416.3095 808.00341 116.38 - Sant_1337 Predicted product Sant_0424 bfrA Gene name Locus Table 3.1. (Continued) 42 Gene name Sant_3443 yhbT - 69.983 43.503 Endo/exonucl./phosph 101.21365 105.2521 270.60492 43.226 73.421 at. fam. protein Sant_1580 164.34692 160.6974 301.09561 64.007 90.965 199.55 2;5-diketo-D-gluconate 648.36862 697.2949 1046.212 168.75 456.12 reductase A Sant_3594 dkgA Putative sterol transferase 3.7828 hypothetical protein 17.035961 13.15651 13.339679 4.9876 3.8985 - 743.33 Sant_2442 1917.0466 2039.259 4524.0569 722.37 1404.1 118.21 Aldehyde dehydrogenase family 663.40035 655.0062 1486.4214 170.41 560.08 protein Putative sigma-54 modulation protein - 26.48 86.181919 55.44528 133.39679 38.238 27.289 Putative acetyltransferase 44.448 ct ct ct ct ct ct OHHL NOOHHL NOOHHL NOOHHL OHHL OHHL 1 3 2 1 3 2 TRNA-dihydrouridine 122.25807 120.2881 194.37818 43.226 61.726 synthase A Predicted product Sant_3174 raiA Sant_0106 Sant_3442 yhbS Sant_1298 yfcZ Locus Table 3.1. (Continued) log2 Fold change 1.2913438 1.2997654 1.2688344 0.001421 1.2660344 NA 0.003826 1.2806439 NA NA 0.014076 1.3078212 0.002476 1.3079416 0.000470 1.3319566 padj 43 707.4934 639.9702 996.6646 262.679 Pyruvate kinase Sant_2049 pykF ct OHHL 1 padj log2 Fold change 270.295 345.185 0.000241 1.265886 ct OHHL 2 - - - - Sant_r0474 Sant_r0265 Sant_3780 Sant_2233 Sant_4024 Sant_1359 menB 1.240837 hypothetical 302.6388 320.455 514.5305 87.2825 233.2594 83.2226 0.009732 1.220188 protein Put.3-oxoacid CoA-transf.;β 49.10365 62.02354 78.13241 14.1315 36.38587 14.1857 0.009807 1.232437 subunit NA 661.3961 888.0643 1255.835 332.505 324.8738 402.873 0.000639 1.241379 722.5252 873.968 1269.175 361.599 336.5693 390.579 0.000385 1.244345 157.3321 121.2278 165.7932 57.3571 50.03057 60.5255 0.000471 1.245765 MaoC domain protein 48.10154 31.95152 47.64171 6.65009 23.39092 12.2942 dehydratase - - Naphthoate synthase Universal Sant_0199 uspA stress protein 797.6838 925.6543 2073.367 267.666 736.1641 269.528 0.012748 1.250314 A ct OHHL 3 ct ct ct NOOHHL NOOHHL NOOHHL 3 2 1 Predicted product Locus Gene name Table 3.1. (Continued) 44 hypothetical protein 104.22 51.6863 66.6984 - Sant_r3942 Sant_r0476 Sant_2166 padj Urocanate hydratase - - log2 Fold change 1.2082931 1.217225 186.3935 212.384 278.2276 73.151 1.181864 118.254 73.7655 0.0023335 1.1768695 692.4617 831.679 1280.609 361.599 315.128 431.244 0.0016107 515.0873 590.163 901.3812 265.173 237.158 292.225 0.0011303 1.1859228 106.559 28.3713 0.0130996 1.1972223 74.721 19.8599 0.0331567 1.2068822 26.6004 33.7869 21.7514 0.006119 50.707 - Sant_2570 ct OHHL 1 6phosphofructo 748.5802 842.956 1863.744 379.055 451.575 452.05 0.0040782 kinase PTS sys. agaW NAG-spec. IIC 166.3511 139.083 213.4349 component - Sant_P0204 Sant_0023 pfkA Sant_3982 ct OHHL 2 104.8118 16.6252 38.3351 20.8056 0.0096058 1.2181457 56.11846 56.385 2-dehyd.-3deoxygluc. aldolase ct OHHL 3 19.119 garL Sant_3269 ct ct ct NOOHHL NOOHHL NOOHHL 3 2 1 Predicted product Stress induced 57.12057 110.891 184.8498 protein Gene name Locus Table 3.1. (Continued) 45 - - Sant_r0930 Sant_2232 Sant_3367 leuB Gene name Locus ct OHHL 3 ct OHHL 2 ct OHHL 1 padj log2 Fold change 700.4786 850.4743 1200.571 344.1424 309.9296 442.593 0.0013958 1.1765077 ct ct ct NOOHHL NOOHHL NOOHHL 3 2 1 3-isopropylmalate dehydrogenase 60.12692 44.16828 121.9628 29.92543 28.5889 24.5885 0.0135024 1.1652174 Putative coenzyme A 51.10788 79.8788 89.56642 25.76912 35.73612 21.7514 0.0067061 1.1747892 transferase - Predicted product Table 3.1. (Continued) 46 Figure 3.2. Transcriptomic (RNA-Seq) analysis of genes mediating QS in S. praecaptivus. (A) Heat map showing changes in expression of the top 30 (most differentially expressed) genes in S. praecaptivus in response to OHHL. The asterisks in the boxes on the left indicate statistically significant levels of differential expression. The matrix on the right-hand side of the panel shows the status of genes in Sodalis-allied insect symbionts that are closely related to S. praecaptivus. (B) Chitinase activities of WT and mutant S. praecaptivus strains, in the presence or absence of OHHL. All samples were assayed in triplicate and error bars show standard deviations. Ordinary one way ANOVA comparisons between genotypes: ΔypeI vs. ΔypeI supplemented with 10 µg/ml OHHL, WT vs. ΔypeI, ΔypeR vs. ΔyenR and ΔypeR vs. ΔypeRyenR, all yield P <0.0001; ΔyenR vs. ΔypeR ΔyenR double mutant yields P = 0.0006. 47 - - regC - - Sant_1984 Sant_P0292 Sant_1090 Sant_P0288 Sant_2857 Sant_3640 Sant_1258 Sant_2856 50.346 47.652 37.226 196.047 197.112 14.917 13.766 18.613 85.0205 113.023 162.56 6.27E-27 -1.67433 78.356 8.45E-19 -1.70044 D-isomer specific 2hydroxyacid dehydrogenase NADbinding protein 35.429 36.004 28.85 115.028 156.424 100.576 1.57E-15 -1.42944 Peptidase M60 vir. enh. 116.54 111.19 84.689 400.096 357.153 295.882 6.60E-31 -1.54172 protein Prophage P2 protein Putative phage lysozyme 158.5 Metallo phosphoesterase 146.13 112.61 593.143 702.552 550.831 1.42E-60 -1.94044 27.038 26.474 30.711 173.042 207.058 126.305 8.71E-30 -1.95585 Put. chitin-binding domain 3 protein log2 Fold change 251.73 155.66 138.67 1803.43 2189.03 1569.46 1.81E-26 -2.09142 padj 1902.77 3.14E-36 -2.28258 ctYenR 1 Fibronectin t. III dmn.cng. protein 2807.5 ctYenR 2 324.45 247.79 208.47 2416.58 ctYpeR ctYpeR ctYpeR ctYenR 3 2 1 3 Putative exochitinase product Note: Ranked according to log2fold change in transcript numbers (first hundred results). Three biological replicates were performed for each strain and the columns containing count data are labeled ctYenR1-3 (for ΔyenR strain) and ctYpeR1-3 (for ΔypeR) Gene name Locus Table 3.2. Transcriptomic comparison of ΔyenR and ΔypeR mutant strains. 48 - Sant_2803 Sant_ps0811 - Putative chitinase hypothetical protein product - Sant_1963 Sant_0813 Sant_0273 Sant_P0204 -1.22704 54.075 50.829 45.602 183.04 156.42 93.559 2.12E-05 -0.94116 hypothetical protein 86.542 6.45E-07 -0.95681 -0.9911 -1.09818 34.496 42.358 33.503 79.019 99.46 NA NA hypothetical protein 47.949 14.89 58.014 57.868 44.441 24.356 23.266 67.016 88.61 32.573 135.03 92.227 105.25 3.63E-10 -1.16951 NA 22.376 16.943 15.85 29.65 -1.33635 log2 Fold change 30.711 230.06 197.11 147.36 6.58E-10 -1.28903 NA padj Protein gp55 Chitinase 49.77 13.96 79.019 54.251 54.966 25.173 14.825 12.098 62.015 85.898 53.797 55.94 9.3233 18.002 ctYpeR ctYpeR ctYpeR ctYenR ctYenR ctYenR 3 2 1 3 2 1 pirB JHE-like toxin 49.414 - Sant_0455 Sant_2443 Gene name Locus Table 3.2. (Continued) 49 25.173 18.002 18.613 53.013 48.83 39.763 1171.9 Putative intracellular protease/amidase Cpn10 chaperonin GroES Sant_P012 6 Sant_3402 dnaK Sant_3551 groES 1160.5 1875.5 1880 Molecular chaperone 1183.1 1301.4 1431.3 1991.5 2165 1149 64.331 70.949 74.452 108.03 137.4 Cysteine synthase A -0.75822 2553 4.62E-15 -0.73487 2206.8 2.38E-21 -0.74548 NA 153.2 1.30E-05 -0.75824 1616.2 2.08E-20 -0.78017 Sant_1243 cysK - -0.91455 log2 Fold change 326.29 7.17E-13 -0.83279 832.57 778.32 874.81 1374.3 1392 368 Heat shock protein HSP90 - Sant_0881 NA padj 31.642 76.018 92.23 83.034 1.75E-05 -0.84331 40.24 43.82 Ser/Thr protein phosphatase domain protein Sant_2972 htpG - Sant_3874 14.825 16.752 44.011 47.92 36.254 Putative phage-related transmembrane 12.12 protein ctYpeR ctYpeR ctYpeR ctYenR ctYenR ctYenR 3 2 1 3 2 1 194.86 200.14 184.27 389.09 - Sant_0812 product Putative glycosidase Gene name Locus Table 3.2. (Continued) 50 Gene name - groEL pirA - ompC gmhA - Locus Sant_ps2409 Sant_3550 Sant_ps2804 Sant_2444 Sant_P0291 Sant_1375 Sant_3151 Sant_2389 log2 Fold change 23.39 NA NA -0.69282 -0.69293 -0.70818 123.78 187.05 197.11 205.83 6.86E-06 -0.65246 Lytic transglycosyla 48.4813 51.888 52.116 93.022 78.664 99.407 0.00105 -0.64567 se catalytic Phosphohepto 110.948 118.6 se isomerase 2988.13 2775.5 2867.3 4369.1 4312.1 5196.1 3.63E-17 -0.65926 17.18 hypothetical 5.59399 6.3537 6.5145 21.005 protein 22.22 NA 5699.2 5983.1 8993.2 9345.7 10433 1.30E-25 -0.71979 9.32332 10.589 12.098 29.007 29.838 25.729 5525 17.18 Outer membrane protein C padj 44.752 50.829 48.394 89.021 101.27 81.865 0.00019 -0.72184 ctYpeR ctYpeR ctYpeR ctYenR ctYenR ctYenR 3 2 1 3 2 1 JHE-like toxin 7.45866 4.2358 7.4452 24.006 - Cpn60 chaperonin GroEL - product Table 3.2. (Continued) 51 log2 Fold change clpB Sant_0925 metN Sant_3170 Sant_1266 nupC lon 234.03 213.12 349.08 343.59 354.36 2.54E-07 -0.5992 950.8 1.56E-09 -0.60947 428.58 491.19 4.28E-08 -0.61063 139.6 228.05 230.57 243.25 5.82E-05 -0.56346 322.59 318.74 327.59 444.11 503.63 532.12 1.17E-07 -0.57079 221.9 Put. ABC transport. 169.68 144.02 ATP-binding protein ATP-dependent chaperone protein Nucl. transport protein 278.77 273.21 289.43 409.1 DNA-binding ATP514.65 517.82 607.71 774.19 847.22 dependent protease 192.73 207.53 305.07 294.76 346.17 3.55E-07 -0.63677 Sant_2998 181.8 Heat shock protein Carbamoyl phosphate synthase small subunit Sant_1085 grpE carA Sant_3390 padj Put. LPS biosynth. 112.81 124.96 107.96 173.04 188.97 210.51 2.19E-05 -0.63705 glycosyltransferase ctYpeR ctYpeR ctYpeR ctYenR ctYenR ctYenR 3 2 1 3 2 1 Extracell. polysacch. 148.24 132.37 136.81 217.05 230.57 229.22 6.06E-06 -0.62373 pyruvyl transferase - Sant_2430 product Sant_1450 wcaK Gene name Locus Table 3.2. (Continued) 52 prs - Sant_2116 Sant_0719 - Sant_1921 fnr ibpB Sant_1928 Sant_0091 Sant_2100 nadE ftnA Sant_1707 Sant_1334 ackA Gene name Locus log2 Fold change 99.024 92.227 88.881 0.005286 -0.55314 Heat shock protein B 67.128 57.183 67.007 101.02 98.556 101.75 0.006981 -0.52842 Fumarate/nitrate reduction transcriptional 229.35 245.67 214.05 339.08 349.02 335.64 8.54E-06 -0.53087 regulator 118.41 109.07 107.96 180.04 161.85 170.75 0.000698 -0.53366 NH(3)-dependent NAD(+) synthetase -0.54471 189.55 211.26 290.07 311.94 326.29 1.83E-05 -0.53729 NA 216.3 21.444 12.707 17.682 36.009 29.838 35.085 961.23 972.11 911.11 1356.3 1333.7 1537.9 2.48E-11 -0.54862 57.7 hypothetical protein Ferritin Acetate kinase (Acetokinase) padj 491.34 475.46 474.63 717.17 700.74 749.65 3.70E-10 -0.56065 ctYpeR ctYpeR ctYpeR ctYenR ctYenR ctYenR 3 2 1 3 2 1 DeoR family 58.737 57.183 transcriptional regulator Putative ribosephosphate pyrophosphokinase product Table 3.2. (Continued) 53 Gene name pta artI - apt pgi Sant_0323 Sant_2976 Sant_3883 Sant_3011 yajQ Sant_2660 Sant_0489 fimC1 Sant_1333 Sant_2492 yceA Locus Glucose-6-phosphate isomerase 151.97 137.662 164.725 226.05 Adenine phosphoribosyl transferase 423.279 419.341 377.843 538.13 622.08 617.49 9.16E-07 -0.511787 235.07 0.00035 -0.512475 44.752 44.4756 39.0873 70.017 68.718 67.831 0.021079 -0.517271 5-carboxymethyl-2hydroxymuconate deltaisomerase 216.1 104.421 113.307 108.886 157.04 179.03 156.71 0.001271 -0.520234 Putative nucleotidebinding protein Arginine ABC transporter 238.677 223.437 221.494 309.07 341.78 362.54 1.98E-05 -0.521632 periplasmic component 87.6392 95.3048 99.5794 153.04 131.11 148.53 0.002166 -0.523663 Fimbrial chaperone protein log2 Fold change 1557.93 1428.51 1368.05 2011.5 2066.1 2273.5 3.86E-11 -0.524189 padj Phosphate acetyltransferase ctYpeR ctYenR ctYenR ctYenR 1 3 2 1 167.82 162.018 161.002 242.06 226.95 268.98 0.000132 -0.52724 ctYpeR ctYpeR 3 2 Rhodanese domain protein product Table 3.2. (Continued) 54 - sfcA lysP Sant_3766 Sant_1437 Sant_1419 dan ybjX - Sant_3073 Sant_3639 Sant_2512 Sant_2332 Sant_2718 glnH Gene name Locus 151 157.28 250.06 216.1 NA -0.5009 -0.50126 Anaerobic C4dicarboxylate transporter Virulence factor VirK 158.5 119.66 472.69 457.46 614.15 653.73 660.76 5.38E-07 -0.49241 158.21 196.05 206.15 244.42 0.001794 -0.49142 416 2;5-diketo-D-gluconic acid 39.158 46.593 45.6018 64.015 84.993 58.475 0.032552 -0.49566 reductase A 41.023 33.886 38.1566 64.015 56.964 61.983 NA 268.98 0.000439 -0.50131 Cysteine ABC transporter periplasmic substrate 35.429 24.356 38.1566 52.013 45.209 67.831 binding protein N-acyl-D-amino-acid deacylase log2 Fold change 196.47 0.004819 -0.50651 padj 194.86 176.84 183.338 288.07 245.03 284.19 0.000187 -0.50351 87.639 109.07 132.152 153.04 ctYpeR ctYpeR ctYpeR ctYenR ctYenR ctYenR 3 2 1 3 2 1 Lysine-specific permease175.28 165.19 associated protein Malate dehydrogenase Alcohol dehydrogenase GroES domain protein product Table 3.2. (Continued) 55 Gene name prlC ahpC ptsH gpt thiM efeU - Locus Sant_0488 Sant_0195 Sant_3035 Sant_1242 Sant_3146 Sant_1176 Sant_1399 Sant_1541 326.32 343.1 308.98 470.113 438.53 padj -0.491 822.32 797.38 690.54 1053.25 1064.226 1177.7 1.44E-07 -0.48811 909.02 816.44 867.36 1169.28 1139.274 1406.9 3.18E-07 -0.4912 NA -0.48042 63.399 79.421 55.839 101.024 90.41855 109.93 0.019387 -0.48322 Hydantoinase/520.511 19.061 26.058 40.0096 31.64649 42.102 oxoprolinase Iron permease FTR1 Hydroxyethylthiaz 50.346 48.711 62.353 81.0195 81.37669 86.542 0.024559 -0.48465 ole kinase Xanthine-guanine phosphoribosyl 154.77 168.37 182.41 240.058 247.7468 245.59 0.000455 -0.48583 transferase Phosphocarrier protein Putative peroxidase log2 Fold change 499.37 6.79E-06 -0.49141 ctYpeR ctYpeR ctYpeR ctYenR ctYenR ctYenR 2 3 2 1 3 1 Oligopeptidase A 320.72 312.39 343.41 428.103 462.0388 519.25 1.32E-05 Fimbrial protein product Table 3.2. (Continued) 56 Gene name - - Sant_1983 Sant_1576 TonB-dependent siderophore receptor hypothetical protein padj log2 Fold change 90.022 48.826 51.458 NA -0.47291 97.652 128.64 0.02375 -0.47861 138.92 147.193 155.42 208.05 188.975 242.09 0.00222 -0.46846 33.564 31.7683 31.642 55.013 85.62 193.93 183.197 223.36 256.06 292.052 321.61 0.00054 -0.47961 ctYpeR ctYpeR ctYpeR ctYenR ctYenR ctYenR 3 2 1 3 2 1 Heat shock protein 62.466 58.2418 A Fumarate hydratase product - Sant_1609 Sant_2142 Sant_4066 xanP 108.15 101.658 142.39 175.04 153.712 185.95 0.00756 -0.46419 hypothetical protein 42.887 42.3577 45.602 78.019 58.7721 64.322 0.05143 -0.45954 Putative carbon110.95 99.5406 95.857 154.04 146.478 143.85 0.00726 -0.46298 nitrogen hydrolase Uracil-xanthine permease Thioredoxin Sant_2961 ybbN domain-containing 131.46 135.545 164.72 189.05 204.346 230.39 0.00296 -0.46487 protein ibpA Sant_0090 Sant_2005 fumC Locus Table 3.2. (Continued) 57 Gene name - - Sant_0557 Sant_2522 hypothetical protein padj log2 Fold change 398.8 0.00024 NA -0.45376 -0.45648 70.17 36.295 44.0106 63.293 47.949 41.955 46.593 48.394 65.0157 70.526 29.65 -0.44431 0.057115 -0.44397 NA 58.737 48.711 46.532 88.0212 61.485 84.204 0.051045 -0.45079 -0.4513 115.4 149.036 147.38 167.24 0.008236 -0.45369 267.1 434.105 349.02 45.61 471.76 481.82 422.51 622.15 662.77 625.68 5.70E-06 108.15 97.423 296.48 276.38 25.173 24.356 29.781 39.0094 44.305 592.96 655.49 621.67 815.197 859.88 930.92 7.92E-07 -0.45683 ctYpeR ctYpeR ctYpeR ctYenR ctYenR ctYenR 3 2 1 3 2 1 D-isomer specific 2hydroxyacid dehydrogenase 33.564 NAD-binding; inverted Glycine cleavage system transcriptional repressor Exodeoxyribonuclease III Sant_1209 gcvR xthA Sant_1824 hypothetical protein Aromatic amino acid aminotransferase - Sant_1016 Transcriptional regulator; LacI family Acriflavin efflux protein product Sant_2614 aspC - Sant_1613 Sant_2980 acrA2 Locus Table 3.2. (Continued) 58 trmA fcuA Sant_3949 Sant_2912 Sant_3852 dnaJ hslV Sant_3401 Sant_2234 Sant_1180 Sant_3969 Sant_2674 dacC Gene name Locus 432.1 -0.441437 -0.443067 415.93 492.36 0.00012 -0.440526 NA 0.0061 log2 Fold change 32.827 26.058 47.011 47.018 45.61 NA -0.436012 170.617 148.25 122.85 197.05 202.54 223.37 0.00706 -0.435543 30.767 254.527 270.03 307.11 321.08 392.42 451.42 0.00166 -0.438419 147.309 144.02 126.57 194.05 186.26 209.34 0.00492 -0.439417 325.384 309.21 329.45 194.14 23.006 27.126 29.237 216.1 padj ATP-dependent protease 117.474 110.13 104.23 135.03 152.81 184.78 0.01588 -0.433156 subunit Protease/peptidase U32 LacI family transcriptional repressor Chaperone protein D-alanyl-D-alanine carboxypeptidase; Penicillin binding protein TonB-dependent siderophore receptor 14.89 150.106 140.84 122.85 175.04 ctYpeR ctYpeR ctYpeR ctYenR ctYenR ctYenR 3 2 1 3 2 1 Putative phage repressor 14.9173 14.825 CI TRNA (uracil-5-)methyltransferase product Table 3.2. (Continued) 59 Gene name - - - Locus Sant_1645 Sant_3072 Sant_2540 81.02 77.76 87.712 18.613 33.008 31.646 31.576 NA -0.427656 20.12 0.0195 -0.430702 Tail sheath protein 19.579 log2 Fold change 0.0502 -0.432591 padj Alanine racemase domain-containing 123.07 87.892 101.441 135.03 149.19 161.39 protein 53.143 58.242 56.7696 ctYpeR ctYpeR ctYpeR ctYenR ctYenR ctYenR 3 2 1 3 2 1 hypothetical protein product Table 3.2. (Continued) 60 61 Table 3.3. Pairwise log-rank tests on weevil survival. Strain 1 (1) 2 Df (2) Strain 2 Χ P-value Significance ∆ypeI ∆ypeR∆yenR 4.02 1 0.045 ∆ypeI ∆ypeR 127.0 1 1.7e-29 ** ∆ypeI ∆yenR 196.0 1 1.5e-44 ** ∆ypeI WT 231.0 1 3.9e-52 ** ∆ypeR∆yenR ∆ypeR 90.6 1 1.8e-21 ** ∆ypeR∆yenR ∆yenR 135.0 1 3.3e-31 ** ∆ypeR∆yenR WT 151.0 1 1.3e-34 ** ∆ypeR ∆yenR 19.5 1 1.0e-05 ** ∆ypeR WT 133.0 1 8.1e-31 ** ∆yenR WT 92.1 1 8.1e-22 ** WT ∆ypeI-1 + WT 3.2 1 0.074 WT ∆ypeR + WT 104.0 1 2.3e-24 ** WT ∆ypeI-1 192.0 1 1.1e-43 ** ∆ypeI-1 + WT ∆ypeR + WT 34.7 1 3.7e-09 ** ∆ypeI-1 + WT ∆ypeR 51.0 1 9.1e-13 ** ∆ypeI-1 + WT ∆ypeI 112.0 1 4.1e-26 ** ∆ypeR + WT ∆ypeR 2.16 1 0.14 ∆ypeR + WT ∆ypeI 54.1 1 1.9e-13 ** ∆ypeR ∆ypeI 90.8 1 1.6e-21 ** WT ∆cpmAJ 0.015 1 0.9 WT ∆ypeI ∆pirA 147.0 1 9.3e-34 ** WT ∆ypeI ∆pirB 170.0 1 5.8e-39 ** WT ∆ypeI ∆regC 147.0 1 8.7e-34 ** WT ∆ypeI ∆cpmAJ 227.0 1 2.7e-51 ** ∆cpmAJ ∆ypeI ∆pirA 158.0 1 2.7e-36 ** ∆cpmAJ ∆ypeI ∆pirB 191.0 1 2.1e-43 ** ∆cpmAJ ∆ypeI ∆regC 162.0 1 3.6e-37 ** ∆cpmAJ ∆ypeI 230.0 1 5.2e-52 ** ∆cpmAJ ∆ypeI ∆cpmAJ 226.0 1 4.2e-51 ** ∆ypeI ∆pirA ∆ypeI ∆pirB 3.0 1 0.083 ∆ypeI ∆pirA ∆ypeI ∆regC 1.33 1 0.25 ∆ypeI ∆pirA ∆ypeI 105.0 1 1.3e-24 ** ∆ypeI ∆pirA ∆ypeI ∆cpmAJ 97.7 1 4.9e-23 ** ∆ypeI ∆pirB ∆ypeI ∆regC 0.229 1 0.63 (3) 62 Table 3.3. (Continued) Strain 1 (1) 2 Χ ∆ypeI ∆pirB ∆ypeI 222.0 1 3.2e-50 ** ∆ypeI ∆regC ∆ypeI 145.0 1 2.6e-33 ** ∆ypeI ∆regC ∆ypeI ∆cpmAJ 134.0 1 5.0e-31 ** ∆ypeI ∆ypeI ∆cpmAJ 0.399 1 0.53 ∆ypeR ∆ypeI ∆pirA 1.42 1 0.23 ∆ypeR ∆ypeI ∆pirB 0.112 1 0.74 ∆ypeR ∆ypeI ∆regC 0.0161 1 0.9 WT ∆ypeI ∆pirAB 169.0 1 1.5e-38 ** ∆ypeI ∆ypeI ∆pirAB 6.73 1 0.0095 ∆ypeR ∆ypeI ∆pirAB 2.56 1 0.11 ∆yenR ∆ypeI ∆pirAB 44.6 1 2.4e-11 ** ∆ypeR∆yenR ∆ypeI ∆pirAB 66.3 1 3.8e-16 ** ∆cpmAJ ∆ypeI ∆pirAB 170.0 1 7.6e-39 ** ∆ypeI ∆pirA ∆ypeI ∆pirAB 0.152 1 0.7 ∆ypeI ∆pirB ∆ypeI ∆pirAB 4.94 1 0.026 ∆ypeI ∆regC ∆ypeI ∆pirAB 2.54 1 0.11 ∆ypeI ∆cpmAJ ∆ypeI ∆pirAB 89.7 1 2.8e-21 ** ∆ypeI∆pirAB ∆regC ∆ypeI ∆pirAB 13.1 1 0.00029 ** WT + ∆ypeI-1 ∆ypeI ∆pirAB 66.4 1 3.7e-16 ** WT + ∆yenR ∆ypeI ∆pirAB 20.9 1 4.9e-06 ** WT + ∆ypeR ∆ypeI ∆pirAB 8.52 1 0.0035 WT ∆ypeI ∆pirAB ∆regC 91.6 1 1.1e-21 ** ∆ypeI ∆ypeI ∆pirAB ∆regC 95.8 1 1.3e-22 ** ∆ypeR ∆ypeI ∆pirAB ∆regC 5.08 1 0.024 ∆yenR ∆ypeI ∆pirAB ∆regC 8.38 1 0.0038 ∆ypeR∆yenR ∆ypeI ∆pirAB ∆regC 55.0 1 1.2e-13 ** ∆cpmAJ ∆ypeI ∆pirAB ∆regC 98.2 1 3.8e-23 ** ∆ypeI ∆pirA ∆ypeI ∆pirAB ∆regC 10.2 1 0.0014 ∆ypeI ∆pirB ∆ypeI ∆pirAB ∆regC 3.46 1 0.063 ∆ypeI ∆regC ∆ypeI ∆pirAB ∆regC 3.98 1 0.046 ∆ypeI ∆cpmAJ ∆ypeI ∆pirAB ∆regC 93.7 1 3.7e-22 ** WT + ∆ypeI-1 ∆ypeI ∆pirAB ∆regC 33.7 1 6.4e-09 ** WT + ∆yenR ∆ypeI ∆pirAB ∆regC 5.4 1 0.02 WT + ∆ypeR ∆ypeI ∆pirAB ∆regC 0.0096 1 0.92 WT WT + ∆yenR 1 5.5e-12 ** 47.5 Df (2) Strain 2 P-value Significance ** ** ** (3) 63 Table 3.3. (Continued) Strain 1 1 2 (1) 2 Df (2) Strain 2 Χ P-value Significance ∆ypeI WT + ∆yenR 57.5 1 3.4e-14 ** ∆ypeR WT + ∆yenR 11.9 1 0.00057 ** ∆yenR WT + ∆yenR 0.0745 1 0.78 ∆ypeR∆yenR WT + ∆yenR 54.0 1 2.0e-13 ** ∆cpmAJ WT + ∆yenR 46.9 1 7.6e-12 ** ∆ypeI ∆pirA WT + ∆yenR 16.4 1 5.2e-05 ** ∆ypeI ∆pirB WT + ∆yenR 13.2 1 0.00028 ** ∆ypeI ∆regC WT + ∆yenR 11.9 1 0.00056 ** ∆ypeI ∆cpmAJ WT + ∆yenR 87.3 1 9.4e-21 ** WT + ∆ypeI-1 WT + ∆yenR 13.3 1 0.00027 ** WT + ∆ypeR WT + ∆yenR 4.22 1 0.04 ∆ypeI ∆pirB ∆ypeI ∆cpmAJ 210.0 1 1.4e-47 ** (3) ∆ypeI indicates the ∆ypeI::Spc allele, ∆ypeI-1 indicates the ∆ypeI::Kan allele. 3 df: degrees of freedom. ** indicates p-value < 0.01, of pairwise log-rank tests Figure 3.3. Weevil movement following injection of S. praecaptivus WT and ΔypeI strains. Histograms depict the average time taken by a group of weevils to complete a movement assay, on days 9, 10, 11 and 12 following injection with WT and ΔypeI strains of S. praecaptivus. Any weevils that could not complete the movement assay within the total observation time of 100 seconds were binned into the >95 second category. Figure 3.4. Weevil survival following injection of WT and mutant S. praecaptivus (A) Inactivation of genes involved in the S. praecaptivus QS system results in weevil killing. Note that the "+" symbol indicates injection of equal numbers of the WT and respective mutant strain. The WT strain complements the killing phenotype of the ΔypeI mutant strain. (B) Highlighting the role of the response regulators YpeR and YenR in weevil killing. Inactivation of both YpeR and YenR in a double mutant strain yields the most potent killing phenotype. (C) Identification of genes that mediate weevil killing. Inactivation of pirA, pirB and regC genes mitigates weevil killing in a ΔypeI genetic background. 64 65 Table 3.4. Weevil infection experiments with S. praecaptivus mutant strains 1 S. praecaptivus genotype Injections Median Lifespan (weeks) wild type 151 17 ∆ypeI::Kan 97 1.4 ∆ypeI::Spc 122 1.4 ∆ypeR::Spc 82 3.1 ∆yenR::Spc 156 4.1 ∆ypeR::Gen ∆yenR::Spc 33 1.4 ∆ypeI::Spc ∆(agaR-agaF)::Gen 30 1.4 ∆ypeI::Spc ∆pirB::Gen 89 3.1 ∆ypeI::Spc ∆Sant_1963::Gen 68 1.4 ∆ypeI::Spc ∆Sant_1090::Gen 43 1.4 ∆ypeI::Spc ∆pirAB::Gen 50 3.1 ∆ypeI::Spc ∆Sant_2430::Gen 50 1.4 ∆ypeI::Spc ∆cpmAJ::Gen 74 1.4 ∆ypeI::Spc ∆Sant_1984::Gen 44 1.4 ∆ypeI::Spc ∆regC::Gen 59 3.1 ∆ypeI::Spc ∆Sant_P0288::Gen 31 1.4 ∆ypeI::Spc ∆dmsA1BCD::Gen 32 1.4 ∆ypeI::Spc ∆pirA::Gen 48 3.1 ∆ypeI::Spc ∆Sant_2857::Gen 67 1.4 66 Table 3.4. (Continued) 1 S. praecaptivus genotype Injections ∆ypeI::Spc ∆Sant_P0292::Gen 39 1.4 ∆ypeI::Spc ∆Sant_1963::Gen ∆sant_1984::Kan 33 1.4 ∆ypeI::Spc ∆sant_1962::Gen 39 1.4 ∆ypeI::Spc ∆pirAB::Gen ∆regC::Kan 23 3.5 ∆(agaR-agaF)::Gen 31 >4 ∆pirB::Gen 39 >4 ∆Sant_1963::Gen 31 >4 ∆Sant_1090::Gen 34 >4 ∆pirAB::Gen 31 >4 ∆Sant_2430::Gen 158 >4 ∆cpmAJ::Gen 140 >4 ∆Sant_1984::Gen 25 >4 ∆regC::Gen 31 >4 ∆Sant_P0288::Gen 30 >4 ∆dmsA1BCD::Gen 44 >4 ∆pirA::Gen 34 >4 ∆Sant_2857::Gen 32 >4 ∆Sant_P0292::Gen 64 >4 ∆Sant_1962::Gen 39 >4 wild type + ∆yenR 22 4.1 wild type + ∆ypeR 24 3.1 wild type + ∆ypeI 79 >4 1 Number of weevils injected with the strain Median Lifespan (weeks) 67 Figure 3.5. QS induces growth suppression in S. praecaptivus. Each bacterial strain (label by genotype) was spotted in two positions on the plate. Panel A shows spots placed either distal (left) or proximal (right) to (A) a strip of sterile paper that was impregnated with exogenous OHHL in methanol (left plate) or methanol alone (right plate). Panel B shows spots placed either distal (left) or proximal (right) to a streak of the S. praecaptivus ΔyenR strain maintaining plasmid pCM66 overexpressing the ypeI gene (left plate) or a streak of the S. praecaptivus ΔypeI strain maintaining plasmid pCM66 alone (right plate). The spots highlighted in the red boxes have a 10-fold higher concentration of cells than their counterparts highlighted in blue. 68 Figure 3.6. Inactivation of cpmA relieves growth suppression in S. praecaptivus. Each bacterial strain (labeled according to genotype) was spotted in two positions on the plate, either proximal (left) or distal (right) to a strip of sterile paper that was impregnated with exogenous OHHL in methanol. Note that deletion of cpmA alone relieves QS-mediated growth suppression. 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Then, having grown to a sufficient population density, it would slow down its own growth and be capable of maintaining an asymptomatic infection in the host. This would allow the host organism to retain fitness and serve as a vector to allow the transfer of S. praecaptivus to a wider range of hosts than would be possible with a pathogen that did not curb its virulence. The attenuation of virulence is the most important characteristic in the conversion from parasitism to mutualism, and S. praecaptivus shows evidence of being on just such an evolutionary trajectory. Further work is required to fully reveal all the virulence effector genes and genes repressed to produce the self-growth inhibition that S. praecaptivus uses, and the various levels of regulation these may be subject to.