Investigation of Quorum Sensing-Dependent Gene

J. Microbiol. Biotechnol. (2014), 24(12), 1609–1621 http://dx.doi.org/10.4014/jmb.1408.08064 Research Article Review jmb

Investigation of Quorum Sensing-Dependent Gene Expression in Burkholderia gladioli BSR3 through RNA-seq Analyses S Sunyoung Kim1, Jungwook Park1, Okhee Choi2, Jinwoo Kim2, and Young-Su Seo1*

1Department of Microbiology, Pusan National University, Busan 609-735, Republic of Korea 2Division of Applied Life Science and Institute of Agriculture and Life Science, Gyeongsang National University, Jinju 660-701, Republic of Korea

Received: August 26, 2014 Revised: September 12, 2014 The plant pathogen Burkholderia gladioli, which has a broad host range that includes rice and Accepted: September 13, 2014 onion, causes bacterial panicle blight and sheath rot. Based on the complete genome sequence First published online of B. gladioli BSR3 isolated from infected rice sheaths, the genome of B. gladioli BSR3 contains September 15, 2014 the luxI/luxR family of genes. Members of this family encode N-acyl-homoserine lactone

*Corresponding author (AHL) quorum sensing (QS) signal synthase and the LuxR-family AHL signal receptor, which Phone: +82-51-510-2267; are similar to B. glumae BGR1. In B. glumae, QS has been shown to play pivotal roles in many Fax: +82-51-514-1778; E-mail: [email protected] bacterial behaviors. In this study, we compared the QS-dependent gene expression between B. gladioli BSR3 and a QS-defective B. gladioli BSR3 mutant in two different culture states (10 and 24 h after incubation, corresponding to an exponential phase and a stationary phase) using RNA sequencing (RNA-seq). RNA-seq analyses including gene ontology and pathway enrichment revealed that the B. gladioli BSR3 QS system regulates genes related to motility, S upplementary data for this toxin production, and oxalogenesis, which were previously reported in B. glumae. Moreover, paper are available on-line only at the uncharacterized polyketide biosynthesis is activated by QS, which was not detected in http://jmb.or.kr. B. glumae. Thus, we observed not only common QS-dependent genes between B. glumae BGR1 pISSN 1017-7825, eISSN 1738-8872 and B. gladioli BSR3, but also unique QS-dependent genes in B. gladioli BSR3.

Introduction that inhabit diverse ecological niches, such as soil, water, plants, and hospitals. This genus includes some members Since the bacterial quorum sensing (QS) system through capable of causing diseases in animals, humans and plants signal molecules was first discovered in Vibrio fischeri [10], [5]. Owing to their clinical importance, many studies of there have been numerous investigations of the mechanisms this genus have focused on human pathogens, including of QS in bacteria [15, 38, 63]. In general, proteobacteria use B. cenocepacia complex (Bcc), B. pseudomallei, and B. mallei, N-acyl-homoserine lactone (AHL) families as a signal which cause lung infections in immunocompromised molecule, and contain gene pairs encoding members of the patients, melioidosis, and glanders, respectively [61]. LuxR-LuxI family, which are AHL signal synthase and Previous studies have shown that the AHL QS circuitry is LuxR-family AHL signal receptors, respectively. When signal widely present in members of the Burkholderia genus and molecules reach threshold levels due to accumulation of plays a pivotal role in the pathogenesis and several the cell population, QS causes bacteria to alter the expression phenotypes, including motility, biofilm formation, production of genes involved in multiple biological processes, such as of siderophore, and proteolytic activities, of some Burkholderia biofilm formation, motility, toxin production, and changes species [9]. in metabolic processes [25, 36, 46, 48, 58]. Burkholderia gladioli was formally classified as Pseudomonas Burkholderia is a genus of proteobacteria with members gladioli, but was later transferred to the Burkholderia genus

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by Yabuuchi et al. [67]. Since it was first identified in rotten secondary metabolites such as uncharacterized polyketide Gladiolus corm in 1913 [54], B. gladioli has been isolated as a were observed in B. gladioli BSR3, but not in B. glumae BGR1. major or opportunistic pathogen from other diseased Overall, our findings will help elucidate the mechanism for plants, fungi, and humans [56]. This pathogen can attack regulation by the QS system in B. gladioli BSR3. Gladiolus, onions, irises, and rice, causing partial or whole decay of plants [21, 54, 66, 69]. Yield loss due to diseases Materials and Methods caused by biotic stress is one of the major concerns in rice production. B. gladioli has been reported to cause bacterial Bacterial Strain, Plasmid, and Culture Conditions panicle blight (BPB) and sheath rot in rice, and in many All bacterial strains and plasmids used in this study are listed in cases, it is isolated from symptomatic rice together with B. Table S1. The B. gladioli strains used included wild-type strain BSR3 glumae [42, 43, 60]. These phylogenetically close species can isolated from rice sheath [53] and a QS-defective BSR3 mutant. produce toxoflavin, a yellow pigment phytotoxin considered B. gladioli was grown at 28°C in Luria-Bertani (LB) medium. Escherichia coli DH5α λpir [29] was used to carry the suicide to be one of the major virulence factors associated with the vector, pVIK112 [20], and E. coli S17-1 λpir was used as a pVIK112 occurrence of disease on rice; however, the severity of vector donor for conjugation experiments [20, 55]. All E. coli strains disease caused by B. glumae is more aggressive [3, 13]. were grown at 37°C in LB medium, whereas Chromobacterium Although B. gladioli-related symptoms in rice may have an violaceum CV026 [37] was grown at 28°C in LB medium. Antibiotics economic impact due to decreased rice quality [13], the (50 or 100 µg/ml kanamycin, 50 or 100 µg/ml rifampicin, and 10 molecular basis for the increased pathogenesis of B. gladioli or 20 µg/ml tetracycline) were added when necessary. relative to B. glumae is not well understood. As described above, these two phytopathogenic Burkholderia Construction of QS-Defective Mutant and Complementation contain the AHL QS system [3]. In B. glumae, the AHL QS Strain system is related to toxin production, motility, protection General and standard techniques for the construction of against visible light, lipase secretion, and oxalate biosynthesis recombinant DNA were used in this study [51]. To generate the [4, 7, 17, 23, 24]. Based on the complete genome sequence of QS-defective B. gladioli BSR3 mutant, bgla_2g11050 that encodes a LuxI family AHL synthase was disrupted by insertion mutagenesis, rice isolate B. gladioli BSR3, it has two AHL QS systems as previously described, using a single crossover recombination of with similar topology to that of B. glumae BGR1 isolated recombinant suicide vector pVIK112 [20]. The internal region of from rice [3, 32, 53]. However, the influences of those AHL bgla_2g11050 was amplified by polymerase chain reaction (PCR) QS systems on B. gladioli are not yet fully understood. using the corresponding primers (Table S2), and then ligated into In this study, we carried out transcriptional profiling of the pGEM-T Easy Vector (Promega). After sequencing to confirm wild-type B. gladioli BSR3 and QS-defective mutant B. gladioli ligation, the cloned vector was cleaved by EcoRI and KpnI, ligated BSR3 through RNA sequencing (RNA-seq) analysis to into pVIK112, and cleaved by the identical restriction enzymes. examine the relationship between AHL QS and physiological Recombinant pVIK112 was transformed into DH5α λpir competent traits of B. gladioli. By analyzing two different culture states cells, which were then cultured overnight in LB medium with (incubation for 10 and 24 h in liquid culture) that represent kanamycin (50 µg/ml). After that, the properly cloned pVIK112 the exponential phase and stationary phase, respectively, plasmids were introduced to S17-1 λpir. Transformed S17-1 λpir we obtained a better understanding of QS-dependent genes. was transferred to B. gladioli BSR3 via biparental mating as follows. Donors were washed twice during the mid-logarithmic Among 7,411 annotated genes in B. gladioli, 448 genes were phase, while recipient cells were washed twice with fresh LB. identified as differentially expressed genes (DEGs) in the Next, the donors were mixed at a 1:1 ratio (500 µl), after which exponential phase, while 1,585 DEGs were observed in the samples were centrifuged. The supernatant was then discarded stationary phase. Our analyses, including gene ontology and the pellet was resuspended in 30 µl of LB broth. Next, (GO) enrichment and the Kyoto Encyclopedia of Genes and samples were spotted onto LB plates and incubated at 28°C Genomes (KEGG) pathway enrichment of these DEGs, overnight. Colonies were then removed and resuspended into revealed that B. gladioli BSR3 exhibits QS-dependent 1 ml of LB broth, after which 100 µl of the suspension was plated physiological features such as flagella motility and toxin on LB agar containing rifampicin (100 µg/ml) and kanamycin production, which are consistent with well-known QS (100 µg/ml). Colonies that grew on the selective plate were collected regulated bacterial behaviors of other Burkholderia, including and confirmed as the COK94 (BSR3 bgla_2g11050::pCOK94) strain B. glumae BGR1. Variations in the metabolic pathway that by PCR using an internal sequence of the pVIIK112 vector and includes inositol degradation and changes in unique upstream sequence of the target gene as primers.

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To generate a construct for complementation of the COK94 Analysis of RNA-seq Data strain, the upstream region and coding sequences of bgla_2g11050 After RNA sequencing, raw reads were mapped to the downloaded were cloned into the pRK415 vector [22]. To accomplish this, DNA reference genome sequences of B. gladioli BSR3 (NCBI BioProject ID fragments were amplified from chromosomal DNA using a pair of PRJNA66301) using the BWA program. SAM files generated by primers, one of which included the KpnI site (5’-AAGGTACCC mapping were converted to BAM files of binary format, and then CGCTTCGCATTTCAAACGA-3’) and another that contained the sorted by chromosomal coordinates using the SAMtools program. EcoRI site (5’-AAAAAGAATTCGTGCAGAACCTCGACCTGAT-3’). Mapped reads per annotated gene (total 7,411 genes) were After purification, the resultant construct was excised by restriction counted by Bam2readcount. The relative transcript abundance enzyme cleavage and ligated with cleaved pRK415 (KpnI/EcoRI), was measured in reads per kilobase of exon per million mapped generating pSLRK01. The constructed plasmid pSLRK01 was sequence reads (RPKM) [39]. Differentially expressed genes were transferred by conjugation from E. coli S17-1 λpir to COK94 as then identified using the DEGseq package in R language [62]. described above, and then plated on LB agar containing kanamycin To analyze DEGs, we conducted a GO enrichment assay using (100 µg/ml) and tetracycline (20 µg/ml). Colonies that grew on the UniProt (http://www.uniprot.org/) database, and then calculated the selective plates were collected and analyzed by colony PCR the p-value. A p-value < 0.01 was considered to indicate significance using primers pRK415_R and a2g_11050_comp_R. for the enriched data. For subsequent analysis of the QS-dependent pathway, DEGs were mapped to the KEGG pathway database Complementation Analysis using the WGET program. The mapped pathway was enriched To monitor the capacity of AHL production by the constructed using a p-value < 0.01. strains, an AHL biosensor system based on C. violaceum CV026 was used. Briefly, 1 ml of overnight culture of C. violaceum CV026 Quantitative Real-Time PCR Analysis that grew in LB broth at 28°C was embedded and mixed To confirm the gene expression patterns analyzed from the thoroughly in 50 ml of liquefied LB agar before pouring on plates. RNA-seq data, quantitative real-time PCR (qPCR) analysis was

Next, 1 ml of supernatants from stationary phase (OD600 > 3.0) performed. The same total RNA samples used to construct the cultures of B. gladioli BSR3, COK94, and COK94comp were used to RNA-seq library were used as the template for synthesis of cDNA extract autoinducers. The equal volume of ethyl acetate was samples. To generate cDNA, a SuperScript III First-Strand utilized, and removed by centrifugal evaporation. The residue Synthesis System (Invitrogen, USA) was used with 1 µg of each was reconstituted in 10 µl of dimethyl sulfoxide (DMSO), after total RNA sample according to the manufacturer’s instructions. which 5 µl of suspension containing extracted autoinducers from The cDNA was then diluted with 190 µl of distilled water and each strain was dropped on the plate and incubated at 28°C for subjected to qPCR using Rotor-gene Q (Qiagen). The reactions 24 h to detect a purple pigment. were conducted by subjecting the samples to initial heating for 10 min at 95°C, then 40 cycles of 95°C for 10 sec, 60°C for 15 sec, RNA Isolation and Library Preparation for RNA-seq and 72°C for 20 sec. All samples were analyzed in triplicate in 25 µl Total RNA was isolated from the cells collected from the reaction mixtures containing 5 µl of diluted cDNA, 2× Rotor-gene cultures of bacteria. To prepare the samples, the wild-type SYBR Green PCR Master Mix (Qiagen), and primers (Table S3). B. gladioli BSR3 and QS-defective mutant were grown in 2 ml of The fold changes were determined by the 2-∆∆Ct method with the LB broth at 28°C with continuous shaking for overnight, after constitutively expressed 16S ribosomal RNA gene as a control for which they were subcultured for 10 or 24h under the same normalization. conditions. Total RNA was then extracted from 4 ml of each bacterial culture using an RNeasy Midi Kit (Qiagen, USA). Residual Phenotypic Analysis genomic DNA contamination was subsequently removed using an QS-dependent phenotypes in B. gladioli BSR3 were investigated RNase-Free DNase kit (Qiagen) according to the manufacturer’s with wild-type and QS-defective mutant strains. Swarming assays protocols. After DNase I treatment, a MICROBExpress bacterial were performed at 28°C on LB plates containing 0.5% Bacto Agar mRNA enrichment kit (Ambion, USA) was used to remove (BD, Franklin Lakes, NJ, USA). Each strain was inoculated into bacterial rRNA from the total RNA sample. 2 ml of LB for 24 h with shaking. Next, 1 ml of stationary-phase RNA-seq libraries were prepared using an Illumina TruSeq culture of each strain was centrifuged, washed twice with fresh RNA sample prep kit (Illumina, USA) according to the standard LB broth, and resuspended in 100 µl of LB broth. Each cell protocol. Sequencing was performed on an Illumina HiSeq2000 suspension (5 µl) was subsequently dropped onto swarming assay instrument using the reagents provided in the Illumina TruSeq PE plates and incubated for 48 h. To determine the production of Cluster kits. The data discussed in this publication have been toxoflavin by each strain, bacterial cells were smeared on LB agar deposited in NCBI’s Gene Expression Omnibus (GEO) and are plates and incubated for 36 h at 28°C. The production of toxoflavin accessible through GEO Series under the accession number by each strain was indicated by the yellow pigment of the culture GSE60490. growing on the plates.

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Results and Discussion expression levels of each gene in four RNA samples used for construction of the RNA-seq library were obtained. For Construction of QS-Defective Mutant of B. gladioli BSR3 comparison with the RNA-seq data, expression levels from

To establish a QS-defective B. gladioli BSR3 mutant strain, qPCR were calculated as log2 fold changes in wild-type we disrupted the bgla_2g11050, which encodes an AHL relative to mutant samples. As shown in Fig. 1, the results synthase. In B. gladioli BSR3, bgla_2g11050 is a putative of each technique were compared by scatter plotting, and ortholog of tofI, a well-known gene encoding AHL synthase correlation coefficients of 0.9982 (Fig. 1A) and 0.9893 of B. glumae BGR1 in the Burkholderia Genome Database, (Fig. 1C) were obtained, indicating that our analysis of the whereas BSR3 has an additional paired AHL synthase RNA-seq data was reliable. (bgla_1p1740) in the genome of the plasmid residing in the polyketide synthesis operon (http://www.burkholderia.com/) Gene Ontology and Pathway Enrichment Analysis of [64]. A QS-defective mutant strain, COK94, was constructed, QS-Dependent Genes and proper disruption of the target gene was confirmed by To obtain a comprehensive understanding of genes PCR verification (data not shown). In addition, the COK94 regulated in QS-dependent manners, we classified DEGs strain was very defective in AHL production as shown by GO enrichment analysis. Overall, 448 DEGs in E phase Fig. S1. Taken together, these findings indicate that the were assigned to 86 GO biological process terms and QS-defective mutant strain of B. gladioli BSR3 was successfully enriched in two terms, 158 GO molecular function terms prepared. were enriched in six terms, and 15 GO cellular components were enriched in two terms based on a p-value < 0.01 Transcriptional Profiling of B. gladioli BSR3 and QS- (Fig. 2A). In the S phase, 1,585 DEGs were assigned to 254 Defective Mutant GO biological process terms and enriched in six terms, 358 To gain insight into the genome-scale gene expression GO molecular function terms and enriched in 10 terms, and patterns regulated by the AHL QS system in B. gladioli 36 cellular components enriched in two terms with the BSR3, we conducted RNA-seq for the wild-type and QS- same criteria (p-value < 0.01) (Fig. 2B). Among these, the defective mutant strains. We acquired the transcriptome of terms associated with nitrogen metabolism (nitrate reductase each strain at two different time points (10 and 24 h), activity; nitrate reductase complex) were identified for representing the exponential phase (OD600 < 1.0; E phase) both conditions. Moreover, the terms connected to the and stationary phase (OD600 > 3.0; S phase), respectively. electron transport system were identified under both RNA-seq was performed using the Illumina Hiseq 2000 conditions in a down-regulated manner, such as cellular system, and four libraries of B. gladioli BSR3 including respiration in the E phase (100% down-regulation), ATP wild-type and mutant strains yielded 35,767,138 to synthesis coupled electron transport (100% down-regulation), 81,669,770 total reads (Table S4). To profile the expression NADH dehydrogenase (ubiquinone) activity (54.55% down- of the genes, we mapped the total reads of the four libraries regulation), and quinone binding (37.50% down-regulation) to the reference genome of B. gladioli BSR3 by the BWA in the S phase (Fig. 2). Conversely, terms signifying histidine program. Overall, 15,590,730 to 25,635,166 reads were able degradation (histidine catabolic process to glutamate and to be mapped (Table S4). formamide/formate) and bacterial motility (cilium or The expression level of genes was quantified based on flagellum-dependent cell motility; motor activity; bacterial- the RPKM [39] value. To identify QS-dependent regulated type flagellum) were only enriched in the S phase with genes, we used the DEGseq package in R language [62], biased regulation (down- or up-regulation) of the genes. with a fold change (FC) calculated from the value of the We also mapped DEGs into the KEGG pathway database wild-type RPKM / mutant RPKM. Consequently, 448 and and identified significant pathways using a cut-off 1,585 genes were identified as DEGs in the E phase and S threshold of p-value < 0.01. The distribution of enriched phase, respectively. An overview and comparison of these pathways under both conditions is shown in Fig. 3. In the E DEGs are provided in Fig. S2. phase, a total of nine pathways were observed, among which two were up-regulated and the rest were down- Validation of RNA-seq Data by qPCR Analysis regulated (Fig. 3A). In the S phase, seven up-regulated and To validate the RNA-seq results, qPCR was carried out four down-regulated pathways were observed. Overall, the using six randomly selected genes (Table S3). The relative results indicated that flagella assembly, starch and sucrose

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Fig. 1. Validation of RNA-seq results using qPCR.

Each log2 ratio of fold changes calculated from qPCR was compared with the log2FC of the RNA-seq data. (A) Correlation of the fold change between RNA-seq (x-axis) and qPCR (y-axis) in the E phase. (B) Log2 fold changes in the E phase measured by qPCR. (C) Correlation of fold changes between RNA-seq (x-axis) and qPCR (y-axis) in the S phase. (D) Log2 fold changes in the S phase measured by qPCR. Error bars represent the standard deviation. metabolism, and inositol phosphate metabolism genes was not observable in the QS-defective strain compared were up-regulated, whereas histidine metabolism genes with wild-type or complemented strains (Fig. 4B). Conversely, were down-regulated (Fig. 3B). putative toxFGHI ortholog genes were only slightly up- regulated in the E phase (1.47- to 5.38-fold) and not QS Controls Biosynthesis of Toxoflavin by B. gladioli identified as DEGs, except for a putative toxF ortholog that Toxoflavin, which is synthesized by different bacteria, encodes an additional protein for typical RND-type three- including B. glumae and B. gladioli, is toxic to various plants, component transport systems [24]. fungi, animals, and bacteria owing to its ability to act as an Taken together, our findings suggest that the biosynthesis effective electron carrier [12]. The molecular bases of of toxoflavin in B. gladioli BSR3 is regulated by the AHL QS toxoflavin biosynthesis and transport are well characterized system in both the E and S phases. However, further in B. glumae [24, 59]. In previous studies, the toxoflavin investigations are needed to identify the regulator genes biosynthesis genes (toxABCDE) and four genes (toxFGHI) induced by autoinducers, even at low cell densities. related to toxoflavin transport were reported to be regulated by the AHL QS system via activation of toxR and toxJ genes QS Controls Bacterial Flagella Motility of B. gladioli in B. glumae BGR1 [24]. In this study, we observed the GO and KEGG pathway enrichment assays revealed that differential expression of putative toxABCDE ortholog genes QS-induced genes belonging to the flagella assembly are and the putative ortholog gene of the transcriptional highly enriched in the S phase (Figs. 2 and 3). We visualized regulator toxJ in B. gladioli BSR3 by the AHL QS system gene expression patterns of the flagella assembly in both (Fig. 4A). In the E phase, these genes were up-regulated QS onset states using a heatmap (Fig. 5), and the regulation 2.77- to 15.41-fold, while they were up-regulated 4.40- to by QS appeared quite different. While all 51 genes were

13.61-fold in the S phase. Moreover, toxoflavin production identified as QS-independent or down-regulated (log2FC =

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Fig. 2. Gene ontology enrichment grouping of QS-dependent genes. Each bar represents the ratio of the number of DEGs (red, up-regulated DEGs; blue, down-regulated DEGs) belonging to the GO terms shown in the y-axis. (A) Results of analysis with DEGs in the E phase. (B) Results of analysis with DEGs in the S phase.

-2.40 to 0.82) in the E phase, over half of the genes were Interestingly, the flhBAFG and fliA genes were all up- differentially up-regulated in the S phase. These findings regulated (log2FC = 1.04 to 2.11) in a QS-dependent manner are consistent with the regulatory mechanism of the AHL during the S phase (Fig. 5). These genes are known to be co- QS system, which is cell density dependent. transcribed in B. glumae BGR1 [19], and to have similar In 2007, Kim et al. [23] reported that the AHL QS system organization in both species (Fig. S3). Among these genes, in B. glumae BGR1 induces expression of an IclR-type flhF, which encodes a signal recognition particle-like GTPase, transcriptional regulator, qsmR, which subsequently activates is involved in the regulation of flagella formation and transcription of the flhDC genes, encoding a master flagella motility in many bacteria [6, 40, 44, 50]. Moreover, the regulator [23]. However, in our analysis, the homolog of regulation of flhF by a QS master regulator in Vibrio vulnificus qsmR, bgla_1g11630 (identities = 88%), or the flhDC genes was recently reported [27]. in B. gladioli BSR3 were QS-independently expressed. Based on the results of previous studies and our finding

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Fig. 3. Enriched QS-dependent pathways. The pathway name based on the KEGG database is shown in the y-axis, and the ratio of the number of DEGs in each pathway is shown in the x- axis. (A) Enriched pathway in the E phase. (B) Enriched pathway in the S phase.

Fig. 4. Organization and expression patterns of putative toxoflavin biosynthesis and transport genes in B. gladioli BSR3. (A) Genetic organization of toxABCDE, toxFGHI, and related regulator toxJ and tofR in B. gladioli BSR3. All genes are putative orthologs of B. glumae identified in the Burkholderia genome database (http://www.burkholderia.com/). Gene expression levels are shown as color variation from yellow to red, corresponding to a range of 0.0 (minimum) to 4.0 (maximum). The genes that did not meet the criteria of p < 0.01 are denoted as non-DEG. (B) The toxoflavin production ability of B. gladioli BSR3 (wild-type), COK94 (mutant), and COK94comp (complemented strain). Yellow pigment was observed on the LB agar plate for all samples except for COK94. The photo was taken 36 h after incubation. of abolished swarming motility in the QS-defective mutant QS Controls myo-Inositol Catabolism of B. gladioli strain (Fig. 5), the AHL QS system of B. gladioli BSR3 myo-Inositol is the most common stereoisomer form of regulates swarming motility by activating the flhBAFG and inositol, a polyol that is characterized as a hydroxylated fliA gene cluster using well-known regulators, such as FlhDC. six-carbon ring and essential for plants as a building block

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and signaling molecule [28, 30, 33]. The catabolism of myo- are associated with myo-inositol catabolism, which yields inosoitol has been studied in various bacteria, especially in acetyl-coenzyme A (CoA) (Fig. 6). In B. gladioli BSR3, nine the gram-positive bacterium Bacillus subtilis, in which its genes are predicted as iol genes (iolA, iolB, iolC, iolD, iolE, catabolic pathway and regulation are well understood [28]. and iolG), two of which are predicted as iolG genes and In B. subtilis, the iolABCDEFGHIJ and iolRS operons are three that are predicted as iolA (mmsA) genes. These known to be involved in inositol utilization [68]. putative iol genes show a tendency for QS-independent In the present study, KEGG pathway analysis revealed expression in the E phase, but a switched pattern in the S that QS-activated genes belonging to the inositol phosphate phase (Fig. 6). Among the three putative iolA genes, only metabolism pathway were highly enriched (43.75%, p- bgla_1g12490 follows the trend of other iol genes. This may value = 5.25E-05) during the S phase. Most of these genes imply that blga_1g12490 is more closely related to the myo- inositol catabolic pathway than the other two putative iolA genes. Based on the results of this study, there are two putative reasons for activation of myo-inositol catabolism by the QS system of B. gladioli BSR3. During plant pathogen infections, carbon metabolism in infected tissues changes [2, 8, 16, 26]. Thus, one can be the utilization of an alternative carbon source when there is high cell density, and the other can be to gain the upper hand in competition with host plants by taking up myo-inositol when B. gladioli BSR3 proliferates. However, the utilization of myo-inositol as the sole carbon source and the mechanism of myo-inositol uptake in B. gladioli BSR3 remain unclear.

QS Controls Polyketide Synthesis of B. gladioli Polyketides are a large family of secondary metabolites that have remarkably diverse structures and functions of medical importance [49]. In bacteria, many species produce polyketides using polyketide synthases (PKSs), which are separated into three types [49]. In the Burkholderia genus, there are several polyketide metabolites, including malleilactone [1], burkholderic acid [14], enacyloxin [35], thailandamide lactone [18], and rhizoxin [47]. To date, there have been several reports of a relationship between the AHL QS system and polyketides, such as up- regulation of pyrrolnitrin biosynthesis in Bcc species [52], the requirement for QS during secretion of enacyloxin by B. ambifaria [35], and induced production of thailandamide lactone in a QS mutant strain of B. thailandensis [18]. Fig. 5. Alteration of flagella motility by QS in B. gladioli BSR3. Although the genes encoding polyketide biosynthesis (A) The gene expression patterns of the flagella assembly that proteins have not been fully characterized in B. gladioli involved genes in the E phase and the S phase were visualized as a BSR3, the genome of B. gladioli BSR3 contains a gene cluster heat map using MeV (Multiple Experiment Viewer), which can be (bgla_2g02250 and bgla_2g02270-300), which appears to downloaded from the public website (http://www.tm4.org/). Each generate an unusual polyketide in B. mallei and B. pseudomallei column of the heat map represents the log FC value of two conditions 2 [45]. Furthermore, including this operon, the majority of with a green-black-red scheme, in which the lower limit is -0.5 and the upper limit is 2.5. (B) The swarming motility test of the B. gladioli BSR3 putative polyketide biosynthesis genes are homologous (wild-type), COK94 (mutant), and COK94comp (complemented with pks genes that encode constituent subunits of bacillaene strain). COK94 showed loss of swarming ability. The photo was taken synthase in Bacullus subtilis [57]. Therefore, we found a 24 h after incubation. number of pks-related genes that are up-regulated by a

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Fig. 6. QS-dependent inositol catabolic pathway in B. gladioli BSR3.

The myo-inositol degradation pathway in B. gladioli BSR3, based on the KEGG database (http://www.kegg.jp/), with minor modifications. The log2 FC of each gene, of which encoded product involved at a specific step of the pathway is visualized as color variation (yellow to red) of arrows with values from a minimum of 0.0 to a maximum of 3.0. E and S represent Log2 FC of E phase and the Log2 FC of S phase, respectively. Abbreviations: a, myo-inositol; b, 2-keto-myo-inositol; c, 3-D-(3,4/5)-trihydroxycyclohexane-1,2-dione; d, 5-deoxy glucuronic acid; e, 2-deoxy-5-keto-D-gluconic acid; f, 2-deoxy-5-keto-D-gluconic acid 6-phosphate; g, dihydroxyacetone phosphate; h, malonate semialdehyde; i, acetyl coenzyme A.

QS-dependent manner in B. gladioli BSR3 (Table 1). The gluconeogenic compounds from acetyl-CoA [11]. In this regulation is tighter in the E phase than in the S phase, pathway, the isocitrate lyase and malate synthase, which indicating that the requirement for this metabolite is are encoded in aceA and aceB, respectively, are required important during the E phase. Moreover, the results of this [65]. With these two enzymes, the glyoxylate cycle bypasses study suggest that these pks genes located on chromosome two oxidative steps in the TCA cycle that evolve CO2, 2 have actual functions, because their transcript abundance enabling the net accumulation of C4-dicarboxylic acids is dramatically altered and they are well conserved in other (e.g., succinate, malate, and oxaloacetate), which is not pathogenic Burkholderia species, excluding B. glumae BGR1 possible with the TCA cycle alone [65]. (data not shown). Taken together, these findings indicate Based on our results, bgla_1g25970 (aceA) is up-regulated that the biosynthesis of this unknown polyketide, which under both conditions, but more tightly during the S phase may resemble bacillaene, is regulated by QS in B. gladioli (log2 FC = 1.71; 4.09), while bgla_1g26010 (aceB) is only up-

BSR3, but not in B. glumae BGR1, due to a lack of this regulated in the S phase (log2FC = 2.91). The finding that operon. QS activates aceAB genes is consistent with previous results of transcriptome analysis of QS regulation in Yersinia pestis QS Controls the Glyoxylate Cycle of B. gladioli [31]. Because microorganisms utilize the glyoxylate cycle The glyoxylate cycle, a variation of the tricarboxylic acid for growth when primary carbon sources are not available (TCA) cycle, permits the synthesis of anaplerotic and [34], the regulation of the glyoxylate cycle by QS may be a

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Table 1. Putative genes related to bacillaene synthesis in B. gladioli BSR3. a b b LocusID Gene name Annotation Log2FC (E ) Log2FC (S ) bgla_1g21570 acpK Acyl carrier protein 0.25 0.23 bgla_1g21580 pksI Polyketide biosynthesis enoyl-CoA hydratase 0.62 0.22 bgla_1g21590 pksH Polyketide biosynthesis enoyl-CoA hydratase 1.28 -0.14 bgla_1g21600 pksE PfaD family protein 0.28 0.19 bgla_1g21610 pksL Polyketide synthase 1.12 0.03 bgla_1g21620 pksN Beta-ketoacyl synthase 1.53 0.62 bgla_1g21630 pksM/pksJ Mixed polyketide synthase/non-ribosomal peptide synthetase 1.46 0.59 bgla_1g21640 pksG Hydroxymethylglutaryl-coenzyme A synthase domain-containing protein 2.12 1.50 bgla_1g21650 pksF Polyketide beta-ketoacyl:acyl carrier protein synthase 2.58 1.53 bgla_1g21660 pksE Malonyl CoA-acyl carrier protein transacylase 2.09 2.11 bgla_1g21670 pksD BatH 1.62 1.01 bgla_2g02200 pksF Polyketide beta-ketoacyl:acyl carrier protein synthase 5.85 2.56 bgla_2g02210 acpK Acyl carrier protein 5.49 2.45 bgla_2g02220 pksI Polyketide biosynthesis enoyl-CoA hydratase 5.76 2.56 bgla_2g02230 pksH Polyketide biosynthesis enoyl-CoA hydratase 5.88 2.26 bgla_2g02240 pksG Hydroxymethylglutaryl-coenzyme A synthase 6.01 2.34 bgla_2g02250 pksE Malonyl CoA-acyl carrier protein transacylase 6.26 2.53 bgla_2g02270 pksL Polyketide synthase, type I 7.83 1.43 bgla_2g02280 pksL Polyketide synthase, type I 7.14 1.04 bgla_2g02290 pksJ Putative polyketide synthase PksJ 7.52 0.52 bgla_2g02300 pksL Putative polyketide synthase 6.58 0.60 bgla_4p2670 Polyketide synthase 2.57 0.34 bgla_4p2680 pksE Protein BatJ 3.69 0.75 bgla_4p2690 pksE Protein BatK 3.31 0.29 bgla_4p2700 pksD Protein BatH 3.37 0.29 aSome gene names are estimated by BLAST match results. bE, E phase; S, S phase. strategy for proliferation in glucose-depleted environments Acknowledgments due to increased cell populations. This up-regulation may also be connected to QS-dependent This research was supported by a National Research oxalate production being an inevitable event in order to Foundation of Korea (NRF) grant funded by the Korean counteract base toxicity during the stationary growth government (MEST) (No. 2013R1A1A2006716). phase of three Burkholderia species (B. glumae, B. mallei, and B. thailandensis) [17]. Among these species, oxalogenesis in References B. glumae is conducted by two QS-induced genes encoding ObcA and ObcB, using oxaloacetate and acetyl-CoA as 1. Biggins JB, Ternei MA, Brady SF. 2012. Malleilactone, a substrates [17, 41]. Given that the putative ortholog genes polyketide synthase-derived virulence factor encoded by the of obcAB (bgla_1g20440-50) in B. gladioli BSR3 are highly cryptic secondary metabolome of Burkholderia pseudomallei group pathogens. J. Am. Chem. Soc. 134: 13192-13195. up-regulated by QS (log2FC = 6.73; 6.83 in the E phase, and 6.16; 6.23 in the S phase, respectively), QS-activated 2. Cangelosi GA, Ankenbauer RG, Nester EW. 1990. Sugars induce the Agrobacterium virulence genes through a periplasmic oxalogenesis using increased oxaloacetate via the glyoxylate binding protein and a transmembrane signal protein. Proc. cycle could be common in Burkholderia species.

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