Structural and Functional Analysis of Phytotoxin Toxoflavin-Degrading Enzyme

Pathogenic bacteria synthesize and secrete toxic low molecular weight compounds as virulence factors. These microbial toxins play essential roles in the pathogenicity of bacteria in various hosts, and are emerging as targets for antivirulence strategies. Toxoflavin, a phytotoxin produced by Burkholderia glumae BGR1, has been known to be the key factor in rice grain rot and wilt in many field crops. Recently, toxoflavin-degrading enzyme (TxDE) was identified from Paenibacillus polymyxa JH2, thereby providing a possible antivirulence strategy for toxoflavin-mediated plant diseases. Here, we report the crystal structure of TxDE in the substrate-free form and in complex with toxoflavin, along with the results of a functional analysis. The overall structure of TxDE is similar to those of the vicinal oxygen chelate superfamily of metalloenzymes, despite the lack of apparent sequence identity. The active site is located at the end of the hydrophobic channel, 9 Å in length, and contains a Mn(II) ion interacting with one histidine residue, two glutamate residues, and three water molecules in an octahedral coordination. In the complex, toxoflavin binds in the hydrophobic active site, specifically the Mn(II)-coordination shell by replacing a ligating water molecule. A functional analysis indicated that TxDE catalyzes the degradation of toxoflavin in a manner dependent on oxygen, Mn(II), and the reducing agent dithiothreitol. These results provide the structural features of TxDE and the early events in catalysis

Burkholderia glumae의 Quorum Sensing과 RpoS에 의한 Universal Stress Protein 유전자의 발현조절

학위논문 (박사)-- 서울대학교 대학원 : 농생명공학부 식물미생물학 전공, 2013. 2. 황인규.대부분의 세균들은 살아가면서 여러가지 환경적인 스트레스 즉, 영양결핍, 온도 스트레스, 산화적 스트레스 그리고 독성 물질등과 같은 스트레스에 노출되어 있다. 이런 환경적인 스트레스를 극복하기 위해서 세균들은 스스로를 방어하기 위한 기작을 가지고 있다. 위와 같은 여러가지 스트레스에 반응하는 유전자 중 하나가 universal stress protein 인 usp 유전자이다. 본 연구는 세균성 벼알마름병을 일으키는 Burkholderia glumae가 quorum sensing (QS)이라는 밀도인식 기작과 stationary phase sigma factor로 알려져 있는 Rpos에 의해서 universal stress protein 유전자의 발현조절 기작과 여러가지 스트레스 중 heat shock stress에 중요한 역할을 한다는 사실을 밝혔다. B. glumae은 신호물질로 N-octanoyl-homoserine lactone (C8-HSL)을 사용하여 cell과 cell 사이에 의사소통을 하는 TofI/TofR QS system을 가지고 있다. tofI는 autoinducer인 C8-HSL를 합성하는 유전자이며, tofR은 이 autoinducer의 receptor 유전자이다. 이 TofR/C8-HSL의 복합체는 toxoflavin의 생합성과 IclR type transcriptional regulator인 qsmR을 조절한다. 우리는 B. glumae의 QS에 의존적인 단백질체 분석을 통하여 universal stress protein들 중 Usp2 단백질을 동정하였다. B. glumae BGR1 전체 genome에서 총 11개의 usp 유전자 (usp1 부터 usp11) 들을 동정하였다. 스트레스 조건에서 실험한 결과 usp1 mutant와 usp2 mutant들은 heat shock stress 후 1시간 내에 죽은 반면, 다른 usp mutants들과 wild-type인 BGR1은 45℃에서 3시간 이상 생존하였다. 모든 usp 유전자들의 발현은 QS에 의해 positive하게 조절되며, 직접적으로는 QsmR에 의해 조절된다. 추가적으로 usp1과 usp2 의 발현은 stationary phase에서 sigma factor인 RpoS에 의해서도 조절된다. 이것은 usp1과 usp2의 promoter region에 RpoS-RNA holoenzyme이 직접 binding하는 것으로 확인을 하였다. usp1의 발현은 37℃에서 28℃ 또는 45℃로 온도 shift에 의해서도 upregulation 되었다. 반면에 usp2의 발현은 온도 스트레스와는 무관하다. 이것은 usp1과 usp2의 발현 조절이 기존에 밝혀져 있는 E. coli의 기작과 다르다는 것을 나타낸다. usp1과 usp2는 heat shock stress 조건에서 B. glumae의 생존에 중요한 역할을 한다는 것을 확인하였다.Burkholderia glumae possesses a quorum sensing (QS) system mediated by N-octanoyl-homoserine lactone (C8-HSL) and its cognate receptor TofR. TofR/C8-HSL regulates the expression of a transcriptional regulator, qsmR. We identified one of the universal stress proteins (Usps), Usp2, from a genome-wide analysis of QS-dependent proteomes of B. glumae. In the whole genome of B. glumae BGR1, 11 usp genes (usp1 to usp11) were identified. Among the stress conditions tested, usp1 and usp2 mutants died 1 h after heat shock stress, whereas the other usp mutants and the wild-type strain survived for more than 3 h at 45°C. The expression of all usp genes was positively regulated by QS, directly by QsmR. In addition, the expression of usp1 and usp2 was dependent on RpoS in the stationary phase, as confirmed by the direct binding of RpoS-RNA holoenzyme to the promoter regions of the usp1 and usp2 genes. The expression of usp1 was upregulated upon a temperature shift from 37℃ to either 28℃ or 45℃, whereas the expression of usp2 was independent of temperature stress. This indicates that the regulation of usp1 and usp2 expression is different from what is known about Escherichia coli. Compared to the diverse roles of Usps in E. coli, Usps in B. glumae are dedicated to heat shock stress.CONTENTS Page ABSTRACT…………………………………………………………………...i CONTENTS………………………………………………………………….iii LIST OF TABLES……………………………………………………………v LIST OF FIGURES………………………………………………………….vi Regulation of Universal Stress Protein Genes by Quorum Sensing and RpoS in Burkholderia glumae ABSTRACT......................................................................................................2 INTRODUCTION............................................................................................3 MATERIALS AND METHODS.....................................................................7 I. Bacterial strains and culture conditions..........................................................7 II. Protein sample preparation for two-deimensional electrophoresis (2-DE) ............................................................................................................7 III. 2-DE and tandem mass spectrometry (MS/MS) analysis………….............8 IV. Multiple-sequence alignment and phylogenetic analysis…………............10 V. DNA construction and mutagenesis………………………………………..10 VI. Reverse transcription (RT)-PCR analysis………………………………...12 VII. Overexpression and purification of B. glumae QsmR and RpoS………..12 VIII. Gel mobility shift assay…………………………………………………13 IX. β-Glucuronidase assay……………………………………………………15 X. Survival tests under heat shock stress ……………………………………..15 XI. Survival under cold shock stress …………………………………………16 XII. Sensitivity to oxidative stress………………………………………........16 XIII. Motility assay…………………………………………………………...16 XIV. UV sensitivity…………………………………………………………...17 XV. Sensitivity to ethanol, acid, and base…………………………………….17 XVI. Long-term survival in the stationary phase……………………………..17 XVII. Sensitivity to mitomycin C and osmotic stress………………………...17 XVIII. Plant Inoculation ...…………………………………………………...18 RESULTS …………………………………………………………………...19 I. Identification of QS-dependent usp genes in B. glumae……………………19 II. Classification of Usps and their phylogenetic relationships………………19 III. Genomic organization of the usp genes of B. glumae……………………..21 IV. Phenotypes of usp mutants under stress conditions………………………22 V. Expression of usp genes is regulated by QsmR…………………………….23 VI. Expression of usp1 and usp2 is dependent on RpoS……………………..24 VII. Expression of usp1 is upregulated by a temperature shift……………….25 VIII. Mutations of usp1 and usp2 retain full virulence……………………….26 DISCUSSION………………………………………………………………..27 LITERATURE CITED…………………………………………………….. 32 ABSTRACT IN KOREAN………………………………………………….77 ACKNOWLEDGEMENT.…………………………………………………79Docto

Rapid and Specific Detection of Apple stem grooving virus by Reverse Transcription-recombinase Polymerase Amplification

Apple stem grooving virus (ASGV) is considered to cause the most economically important viral disease in pears in Korea. The current PCR-based methods used to diagnose ASGV are time-consuming in terms of target detection. In this study, a novel assay for specific ASGV detection that is based on reverse transcription-recombinase polymerase amplification is described. This assay has been shown to be reproducible and able to detect as little as 4.7 ng/μl of purified RNA obtained from an ASGV-infected plant. The major advantage of this assay is that the reaction for the target virus is completed in 1 min, and amplification only requires an incubation temperature of 42°C. This assay is a promising alternative method for pear breeding programs or virus-free certification laboratories

The Quorum Sensing-Dependent Gene katG of Burkholderia glumae Is Important for Protection from Visible Light▿

Quorum sensing (QS) plays important roles in the pathogenicity of Burkholderia glumae, the causative agent of bacterial rice grain rot. We determined how QS is involved in catalase expression in B. glumae. The QS-defective mutant of B. glumae exhibited less catalase activity than wild-type B. glumae. A β-glucuronidase assay of a katG::Tn3-gusA78 reporter fusion protein revealed that katG expression is under the control of QS. Furthermore, katG expression was upregulated by QsmR, a transcriptional activator for flagellar-gene expression that is regulated by QS. A gel mobility shift assay confirmed that QsmR directly activates katG expression. The katG mutant produced toxoflavin but exhibited less severe disease than BGR1 on rice panicles. Under visible light conditions and a photon flux density of 61.6 μmol−1 m−2, the survival rate of the katG mutant was 105-fold lower than that of BGR1. This suggests that KatG is a major catalase that protects bacterial cells from visible light, which probably results in less severe disease caused by the katG mutant

Survey of Major Viruses in Commercial Nursery Trees of Major Pear Cultivars in Korea

Apple chlorotic leaf spot virus (ACLSV), Apple stem pitting virus (ASPV), Apple stem grooving virus (ASGV), and Apple scar skin viroid (ASSVd) are economically important viruses that infect pear tree species worldwide. To evaluate the prevalence of these viruses in Korea, we investigated infection degree of three viruses and one viroid for the commercial nursery trees of the pear cultivars, Niitaka, Chuwhang, Wonwhang, and Whasan in 2017 and 2018. The results showed that the infection ratio of ACLSV, ASPV, ASGV, and ASSVd for the scion of pear cultivar Niitaka was 10%, 45%, 77%, and 50%, respectively. From the scion of pear cultivar Chuwhang, infection ratios of ASPV, ASGV, and ASSVd were found to be 70%, 50%, and 60%, respectively. From the scion of pear cultivar Whasan, infection ratios of ACLSV, ASPV, ASGV and ASSVd were found to be 40%, 60%, 93%, and 20%, respectively. From the root stock of pear cultivar Wonwhang, infection ratios of ACLSV, ASPV, ASGV, and ASSVd showed 28%, 57%, 100%, and 14%, respectively. ASGV had the highest recorded infection rate, and ACLSV was characterized by the lowest infection rate. The mixed infection ratio of Niitaka, Chuwhang, Whasan, and Wonwhang was 45%, 60%, 70%, and 85%, respectively

Development of a Multiplex RT-PCR for the Simultaneous Detection of Three Viruses in Cherry Plants

A multiplex RT-PCR (mRT-PCR) assay was developed for the detection of the recently reported viruses, Cherry virus A (CVA), Little cherry virus 1 (LChV-1), and Little cherry virus 2 (LChV-2), in cherry plants in Korea. Eight sets of primers were designed for each virus and their specificity was tested by using various combinations of mixed primer sets. From the designed primer sets, one combination was selected and further evaluated to estimate the optimum temperature and detection limits of the mRT-PCR. A newly developed mRT-PCR assay was also tested using 20 cherry samples collected in the field. This mRT-PCR assay may be a useful tool for field surveys of diseases and the rapid detection of these three viruses in cherry plants

Biochemical Evidence for ToxR and ToxJ Binding to the tox Operons of Burkholderia glumae and Mutational Analysis of ToxR▿

Burkholderia glumae produces toxoflavin, a phytotoxin with a broad host range, which is a key virulence factor in bacterial rice grain rot. Based on genetic analysis, we previously reported that ToxR, a LysR-type regulator, activates both the toxABCDE (toxoflavin biosynthesis genes) and toxFGHI (toxoflavin transporter genes) operons in the presence of toxoflavin as a coinducer. Quorum sensing regulates the expression of the transcriptional activator ToxJ that is required for tox gene expression. Here, we used gel mobility shift and DNase I protection analyses to demonstrate that both ToxR and ToxJ bind simultaneously to the regulatory regions of both tox operons. ToxR and ToxJ both bound to the toxA and toxF regulatory regions, and the sequences for the binding of ToxR to the regulatory regions of both tox operons possessed T-N11-A motifs. Following random mutagenesis of toxR, 10 ToxR mutants were isolated. We constructed a reporter strain, S6K34 (toxR′A′::Ω toxF::Tn3-gusA34) to evaluate which amino acid residues are important for ToxR activity. Several single amino acid substitutions identified residues that might be important for ToxR binding to DNA and toxoflavin binding. When various toxoflavin derivatives were tested to determine whether toxoflavin is a specific coinducer of ToxR in the S6K34 strain, ToxR, together with toxoflavin, conferred toxF expression, whereas 4,8-dihydrotoxoflavin did so only slightly. With these results, we have demonstrated biochemically that B. glumae cells control toxoflavin production tightly by the requirement of both ToxJ and toxoflavin as coinducers of ToxR