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. Copyright© 2014 by The Korean Society for Microbiology Keywords: Burkholderia glaidioli, gene expression, quorum sensing, RNA-seq and Biotechnology 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 December 2014 ⎪ Vol. 24⎪ No. 12 1610 Kim et al. 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. J. Microbiol. Biotechnol. Quorum Sensing of Burkholderia gladioli BSR3 1611 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.