The lipopolysaccharide (LPS) composed of lipid A, core, and O-antigen is the fundamental constituent of the outer membrane in gram-negative bacteria. This study was conducted to investigate the roles of LPS in Burkholderia glumae, the phytopathogen causing bacterial panicle blight and seedling rot in rice. To study the roles of the core oligosaccharide (OS) and the O-antigen region, mutant strains targeting the waaC and the wbiFGHI genes were generated. The LPS profile was greatly affected by disruption of the waaC gene and slight reductions were observed in the O-antigen region following wbiFGHI deletions. The results indicated that disruption in the core OS biosynthesis-related gene, waaC, was associated with increased sensitivity to environmental stress conditions including acidic, osmotic, saline, and detergent stress, and to polymyxin B. Moreover, significant impairment in the swimming and swarming motility and attenuation of bacterial virulence to rice were also observed in the waaC-defective mutant. The motility and virulence of O-antigen mutants defective in any gene of the wbiFGHI operon, were not significantly different from the wild-type except in slight decrease in swimming and swarming motility with wbiH deletion. Altogether, the results of present study indicated that the LPS, particularly the core OS region, is required for tolerance to environmental stress and full virulence in B. glumae. To our knowledge, this is the first functional study of LPS in a plant pathogenic Burkholderia sp. and presents a step forward toward full understanding of B. glumae pathogenesis.
Background: Panax ginseng Meyer is one of the most valuable medicinal plants which is enriched in anti-microbe secondary metabolites and widely used in traditional medicine. Botrytis cinerea is a necrotrophic fungus that causes gray mold disease in a broad range of hosts. B. cinerea could overcome the ginseng defense and cause serious leaf and root diseases with unknown mechanism. Methods: We conducted simultaneous transcriptomic and metabolomic analysis of the host to investigate the defense response of ginseng affected by B. cinerea. The gene deletion and replacement were then performed to study the pathogenic gene in B. cinerea during ginseng - fungi interaction. Results: Upon B. cinerea infection, ginseng defense responses were switched from the activation to repression, thus the expression of many defense genes decreased and the biosynthesis of antifungal metabolites were reduced. Particularly, ginseng metabolites like kaempferol, quercetin and luteolin which could inhibit fungi growth were decreased after B. cinerea infection. B. cinerea quercetin dioxygenase (Qdo) involved in catalyzing flavonoids degradation and ∆BcQdo mutants showed increased substrates accumulation and reduced disease development. Conclusion: This work indicates the flavonoids play a role in ginseng defense and BcQdo involves in B. cinerea virulence towards the P. ginseng. B. cinerea promotes disease development in ginseng by suppressing of defense related genes expression and reduction of antifungal metabolites biosynthesis.
Bax, a mammalian pro-apoptotic member of the Bcl-2 family induces cell death when expressed in yeast. To investigate whether Bax expression can induce cell death in plant, we produced transgenic Arabidopsis plants that contained murine Bax cDNA under control of a glucocorticoid-inducible promoter. Transgenic plants treated with dexamethasone, a strong synthetic glucocorticoid, induced Bax accumulation and cell death, suggesting that some elements of cell death mechanism by Bax may be conserved among various organisms. Therefore, we developed novel yeast genetic system, and cloned several Plant Bax Inhibitors (PBIs). Here, we report the function of two PBIs in detail. PBI1 is ascorbate peroxidase (sAPX). Fluorescence method of dihydrorhodamine123 oxidation revealed that expression of Bax in yeast cells generated reactive oxygen species (ROS), and which was greatly reduced by co-expression with sAPX. These results suggest that sAPX inhibits the generation of ROS by Bax, which in turn suppresses Baxinduced cell death in yeast. PBI2 encodes nucleoside diphosphate kinase (NDPK). ROS stress strongly induces the expression of the NDPK2 gene in Arabidopsis thaliana (AtNDPK2). Transgenic plants overexpressing AtNDPK2 have lower levels of ROS than wildtype plants. Mutants lacking AtNDPK2 had higher levels of ROS than wildtype. $H_2O_2$ treatment induced the phosphorylation of two endogenous proteins whose molecular weights suggested they are AtMPK3 and AtMPK6. In the absence of $H_2O_2$ treatment, phosphorylation of these proteins was slightly elevated in plants overexpressing AtNDPK2 but markedly decreased in the AtNDPK2 deletion mutant. Yeast two-hybrid and in vitro protein pull-down assays revealed that AtNDPK2 specifically interacts with AtMPK3 and AtMPK6. Furthermore, AtNDPK2 also enhances the MSP phosphorylation activity of AtMPK3 in vitro. Finally, constitutive overexpression of AtNDPK2 in Arabidopsis plants conferred an enhanced tolerance to multiple environmental stresses that elicit ROS accumulation in situ. Thus, AtNDPK2 appears to playa novel regulatory role in $H_2O_2$-mediated MAPK signaling in plants.
Listeria monocytogenes is a foodborne pathogen of considerable genetic diversity with varying pathogenicity. Initially, we found that the strain M7 was far less pathogenic than the strain Lm850658 though both are serovar 4a strains belonging to the lineage III. Comparative genomic approaches were then attempted to decipher the genetic basis that might govern the strain-dependent pathotypes. There are 2,761 coding sequences of 100% nucleotide identity between the two strains, accounting for 95.7% of the total genes in Lm850658 and 92.7% in M7. Lm850658 contains 33 specific genes, including a novel 20K prophage whereas strain M7 has 130 specific genes, including two large prophages (38K and 44K). To examine the roles of these specific prophages in pathogenicity, the 20K and 38K prophages were deleted from their respective strains. There were virtually no differences of pathogenicity between the deletion mutants and their parent strains, although some putative virulent factors like VirB4 are present in the 20K region or holin-lysin in the 38K region. In silico PCR analysis of 29 listeria genomes show that only strain SLCC2540 has the same 18 bp integration hotspot as Lm850658, whereas the sequence identity of their 20K prophages is very low (21.3%). The 38K and 44K prophages are located in two other different hotspots and are conserved in low virulent strains M7, HCC23, and L99. In conclusion, the 20K and 38K prophages of L. monocytogenes serovar 4a strains Lm850658 and M7 are not related to virulence but contribute to genetic diversity.
Proceedings of the Korean Society of Plant Biotechnology Conference
/
2003.04a
/
pp.65-71
/
2003
Bax, a mammalian pro-apoptotic member of the Bcl-2 family, induces cell death when expressed in yeast. To investigate whether Bax expression can induce cell death in plant, we produced transgenic Arabidopsis plants that contained murine Bax cDNA under control of a glucocorticoid-inducible promoter. Transgenic plants treated with dexamethasone, a strong synthetic glucocorticoid, induced Bax accumulation and cell death, suggesting that some elements of cell death mechanism by Bax may be conserved among various organisms. Therefore, we developed novel yeast genetic system, and cloned several Plant Bax Inhibitors (PBIs). Here, we report the function of two PBIs in detail. PBI1 is ascorbate peroxidase (sAPX). Fluorescence method of dihydrorho-damine 123 oxidation revealed that expression of Bax in yeast cells generated reactive oxygen species (ROS), and which was greatly reduced by co-expression with sAPX. These results suggest that sAPX inhibits the generation of ROS by Bax, which in turn suppresses Baxinduced cell death in yeast. PBI2 encodes nucleoside diphosphate kinase (NDPK). ROS stress strongly induces the expression of the NDPK2 gene in Arabidopsis thaliana (AtNDPK2). Transgenic plants overexpressing AtNDPK2 have lower levels of ROS than wildtype plants. Mutants lacking AtNDPK2 had higher levels of ROS than wildtype. $H_2O_2$ treatment induced the phosphorylation of two endogenous proteins whose molecular weights suggested they are AtMPK3 and AtMPK6. In the absence of $H_2O_2$ treatment, phosphorylation of these proteins was slightly elevated in plants overexpressing AtNDPK2 but markedly decreased in the AtNDPK2 deletion mutant. Yeast two-hybrid and in vitro protein pull-down assays revealed that AtNDPK2 specifically interacts with AtMPK3 and AtMPK6. Furthermore, AtNDPK2 also enhances the MBP phosphorylation activity of AtMPK3 in vitro. Finally, constitutive overexpression of AtNDPK2 in Arabidopsis plants conferred an enhanced tolerance to multiple environmental stresses that elicit ROS accumulation in situ. Thus, AtNDPK2 appears to play a novel regulatory role in $H_2O_2$-mediated MAPK signaling in plants.
COP9 signalosome (CSN), which is originally identified as the regulator of the photomorphogenic development in plant, is highly conserved protein complex in diverse eukaryotic organisms. Most eukaryotic CSN complex is composed of 8 subunits, which is structurally and functionally similar to the lid subunit of 26S proteasome and eIF3 translation initiation complex. CSN play important functions in the regulation of cell cycle and checkpoint response by controlling Cullin-Ring E3 ubiquitin ligases (CRL) activities. CSN exhibits an isopeptidase activity which cleaves the neddylated moiety of cullin components. In fission yeast, S-phase cell cycle progression was delayed and the sensitivity to g-ray or UV was increased in CSN1 and CSN2 deletion mutants, indicating that yeast CSN is also involved in the checkpoint regulation. CSN in fungal system more closely resembles that of the higher organisms in the structure and assembly of their components. Functionally, CSN is associated with the regulation of conidiation rhythms in Neurospora crassa and the sexual development in Aspsergillus nidulans. Recent studies also revealed that CSN functions as an essential cell cycle regulator, playing key roles in the regulation of DNA replication and DNA damage response in Aspergillus. Overall, CSN of microorganisms, such as fission yeast and fungi, share functionally common aspects with higher organisms, implying that they can be useful tools to study the role of CSN in the CRL-mediated diverse cellular activities.
By using differential display, we identified one of the genes encoding the multi-subunit complex protein V-ATPase, c subunit gene (ATP6L), and showed alterations of the gene expression by oxidative stresses. Expression of the ATP6L gene in Neuro-2A cells was increased by the treatment with $H_2O_2$ and incubation in hypoxic chamber, implying that the expression of the ATP6L gene is regulated by oxidative stresses. To examine mechanisms involved in the regulation of the gene expression by oxidative stresses, the transcriptional activity of the rat ATP6L promoter was studied. Transcription initiation site was determined by primer extension analysis and DNA sequencing, and promoter of the rat ATP6L and its deletion clones were constructed in reporter assay vector. Significant changes of the promoter activities in Neuro-2A cells were observed in two regions within the proximal 1 kbp promoter, and one containing a suppressor was in -195 to -220, which contains GC box that is activated by binding of Sp1 protein. The suppression of promoter activity was lost in mutants of the GC box. We confirmed by electrophoretic mobility shift and supershift assays that Sp1 protein specifically binds to the GC box. The promoter activity was not changed by the $H_2O_2$ treatment and incubation in hypoxic chamber, however, $H_2O_2$ increased the stability of ATP6L mRNA. These data suggest that the expression of the ATP6L gene by oxidative stresses is regulated at posttranscriptional level, whereas the GC box is important in basal activities of the promoter.
Expression of f3 ~agarase Gene and Catabolite Repression in Escherichia coli by the Promoter of Alginate Lyase Gene Isolated from Marine Pseudomonas sp. Jin, Cheal~Ho, J~Hyeon Park, Jeong-Hyun Han, YoonM Hyeok Chae, Jong~Hee Lee, Jung-Kee Lee!, and In-800 Kong*. Faculty of Food Science and Biotechnology, Pukyong National UniversitYt Pusan 608-737, Korea, llnBioNet Co. 1690-3 Taejon 306-230, Korea - Promoter is a key factor for expression of the recombinant protein. There are many promoters for overexpression of protein in various organisms. The aly promoter of Pseudomonas sp. W7 isolated from marine environment was known to be a constitutive expression promoter of the alginate lyase gene, and it's promoter activity is repressed by glucose in Escherichia coli. To investigate the catabolite repression of the aly promoter ~md association between the promoter mutants, f3 agarase gene, which was also cloned from Pseudomonas sp. W7 was connected to the aly promoter with the sequence the coding 46 N-terminal amino acids ofthe alginate lyase gene. The constructed plasmid was introduced into E. coli and the agarase activity was measured. Fourty six amino acids of the alginate lyase gene was serially deleted using peR to the direction of 5' upstream region and subcloned. The agarase was overexpressed by the aly promoter and the production of agarase was repressed by the addition of glucose into culture media. Fourty six amino acids of alginate lyase did not affect the production of agarase at all. The deletion of a putative stem-loop structure in the aly promoter induced the decrease of f3 -agarase productivity.
Zhang, Chen;Zhou, Zhengfu;Zhang, Wei;Chen, Zhen;Song, Yuan;Lu, Wei;Lin, Min;Chen, Ming
Journal of Microbiology and Biotechnology
/
v.25
no.12
/
pp.2125-2134
/
2015
IrrE is a highly conserved global regulator in the Deinococcus genus and contributes to survival from high doses of UV radiation, ionizing radiation, and desiccation. Drad-IrrE and Dgob-IrrE from Deinococcus radiodurans and Deinococcus gobiensis I-0 each share 66% sequence identity. However, Dgob-IrrE showed a stronger protection phenotype against UV radiation than Drad-IrrE in the D. radiodurans irrE-deletion mutant (ΔirrE), which may be due to amino acid residues differences around the DNA-binding HTH domain. Site-directed mutagenesis was used to generate a Drad-IrrE A184S single mutant, which has been characterized and compared with the ΔirrE mutant complemented strain with Drad-irrE, designated ΔirrE-E. The effects of the A184S mutation following UV radiation and mitomycin C (MMC) shock were determined. The A184S mutant displayed significantly increased resistance to UV radiation and MMC shock. The corresponding A184 site in Dgob-IrrE was inversely mutated, generating the S131A mutant, which exhibited a loss of resistance against UV radiation, MMC shock, and desiccation. qPCR analysis revealed that critical genes in the DNA repair system, such as recA, pprA, uvrA, and ddrB, were remarkably induced after UV radiation and MMC shock in the ΔirrE-IE and A184S mutants. These data suggested that A184S improves the ability against UV radiation and MMC shock, providing new insights into the modification of IrrE. We speculated that the serine residue may determine the efficiency of DNA binding, leading to the increased expression of IrrE-dependent genes important for protection against DNA damage.
The genome of Hantaan virus, the prototype of the hantavirus genus, is composed of three segmented, single stranded negative sense RNA genome. The 5' and 3' termini of the Hantaan virus RNA genome contain noncoding regions (NCRs) that are highly conserved and complementary to form panhandle structures. There are some reports that these NCRs seems to control gene expression and viral replication in influenza virus and vesicular stomatitis virus. In this study, we examined whether NCRs in Hantaan virus playa role in expression of the viral nucleocapsid protein (Np) and foreign (luciferase) gene. The 5' and/or 3' NCR-deleted mutants were constructed and analysed. The Np expression of 5' NCR-deleted clone was similar to that of the clone containing full S genome. In the case of 3' NCR-deleted clone, it showed 40% reduction. To investigate the role of NCR in foreign gene expression, the clones which are replaced ORF of Hantaan viral Np gene with that of luciferase gene were constructed. The results were similar to those of the experiments using Np gene. These results suggest that 3' NCR is more important than 5' NCR in protein expression. To find out a critical region of 3' NCR in protein expression, several clones with a deleted part of 3' NCR were constructed and analyzed. The deletion of the conserved region in 3' NCR showed $20{\sim}30%$ decrease in Np expression. However there were no change in luciferase activities between clones with or without non-conserved region of 3' NCR. These results suggest that the 3' NCR of Hantaan virus S genome, especially conserved region in 3' NCR, plays an important role in the expression of Hantaan viral Np and foreign genes.
본 웹사이트에 게시된 이메일 주소가 전자우편 수집 프로그램이나
그 밖의 기술적 장치를 이용하여 무단으로 수집되는 것을 거부하며,
이를 위반시 정보통신망법에 의해 형사 처벌됨을 유념하시기 바랍니다.
[게시일 2004년 10월 1일]
이용약관
제 1 장 총칙
제 1 조 (목적)
이 이용약관은 KoreaScience 홈페이지(이하 “당 사이트”)에서 제공하는 인터넷 서비스(이하 '서비스')의 가입조건 및 이용에 관한 제반 사항과 기타 필요한 사항을 구체적으로 규정함을 목적으로 합니다.
제 2 조 (용어의 정의)
① "이용자"라 함은 당 사이트에 접속하여 이 약관에 따라 당 사이트가 제공하는 서비스를 받는 회원 및 비회원을
말합니다.
② "회원"이라 함은 서비스를 이용하기 위하여 당 사이트에 개인정보를 제공하여 아이디(ID)와 비밀번호를 부여
받은 자를 말합니다.
③ "회원 아이디(ID)"라 함은 회원의 식별 및 서비스 이용을 위하여 자신이 선정한 문자 및 숫자의 조합을
말합니다.
④ "비밀번호(패스워드)"라 함은 회원이 자신의 비밀보호를 위하여 선정한 문자 및 숫자의 조합을 말합니다.
제 3 조 (이용약관의 효력 및 변경)
① 이 약관은 당 사이트에 게시하거나 기타의 방법으로 회원에게 공지함으로써 효력이 발생합니다.
② 당 사이트는 이 약관을 개정할 경우에 적용일자 및 개정사유를 명시하여 현행 약관과 함께 당 사이트의
초기화면에 그 적용일자 7일 이전부터 적용일자 전일까지 공지합니다. 다만, 회원에게 불리하게 약관내용을
변경하는 경우에는 최소한 30일 이상의 사전 유예기간을 두고 공지합니다. 이 경우 당 사이트는 개정 전
내용과 개정 후 내용을 명확하게 비교하여 이용자가 알기 쉽도록 표시합니다.
제 4 조(약관 외 준칙)
① 이 약관은 당 사이트가 제공하는 서비스에 관한 이용안내와 함께 적용됩니다.
② 이 약관에 명시되지 아니한 사항은 관계법령의 규정이 적용됩니다.
제 2 장 이용계약의 체결
제 5 조 (이용계약의 성립 등)
① 이용계약은 이용고객이 당 사이트가 정한 약관에 「동의합니다」를 선택하고, 당 사이트가 정한
온라인신청양식을 작성하여 서비스 이용을 신청한 후, 당 사이트가 이를 승낙함으로써 성립합니다.
② 제1항의 승낙은 당 사이트가 제공하는 과학기술정보검색, 맞춤정보, 서지정보 등 다른 서비스의 이용승낙을
포함합니다.
제 6 조 (회원가입)
서비스를 이용하고자 하는 고객은 당 사이트에서 정한 회원가입양식에 개인정보를 기재하여 가입을 하여야 합니다.
제 7 조 (개인정보의 보호 및 사용)
당 사이트는 관계법령이 정하는 바에 따라 회원 등록정보를 포함한 회원의 개인정보를 보호하기 위해 노력합니다. 회원 개인정보의 보호 및 사용에 대해서는 관련법령 및 당 사이트의 개인정보 보호정책이 적용됩니다.
제 8 조 (이용 신청의 승낙과 제한)
① 당 사이트는 제6조의 규정에 의한 이용신청고객에 대하여 서비스 이용을 승낙합니다.
② 당 사이트는 아래사항에 해당하는 경우에 대해서 승낙하지 아니 합니다.
- 이용계약 신청서의 내용을 허위로 기재한 경우
- 기타 규정한 제반사항을 위반하며 신청하는 경우
제 9 조 (회원 ID 부여 및 변경 등)
① 당 사이트는 이용고객에 대하여 약관에 정하는 바에 따라 자신이 선정한 회원 ID를 부여합니다.
② 회원 ID는 원칙적으로 변경이 불가하며 부득이한 사유로 인하여 변경 하고자 하는 경우에는 해당 ID를
해지하고 재가입해야 합니다.
③ 기타 회원 개인정보 관리 및 변경 등에 관한 사항은 서비스별 안내에 정하는 바에 의합니다.
제 3 장 계약 당사자의 의무
제 10 조 (KISTI의 의무)
① 당 사이트는 이용고객이 희망한 서비스 제공 개시일에 특별한 사정이 없는 한 서비스를 이용할 수 있도록
하여야 합니다.
② 당 사이트는 개인정보 보호를 위해 보안시스템을 구축하며 개인정보 보호정책을 공시하고 준수합니다.
③ 당 사이트는 회원으로부터 제기되는 의견이나 불만이 정당하다고 객관적으로 인정될 경우에는 적절한 절차를
거쳐 즉시 처리하여야 합니다. 다만, 즉시 처리가 곤란한 경우는 회원에게 그 사유와 처리일정을 통보하여야
합니다.
제 11 조 (회원의 의무)
① 이용자는 회원가입 신청 또는 회원정보 변경 시 실명으로 모든 사항을 사실에 근거하여 작성하여야 하며,
허위 또는 타인의 정보를 등록할 경우 일체의 권리를 주장할 수 없습니다.
② 당 사이트가 관계법령 및 개인정보 보호정책에 의거하여 그 책임을 지는 경우를 제외하고 회원에게 부여된
ID의 비밀번호 관리소홀, 부정사용에 의하여 발생하는 모든 결과에 대한 책임은 회원에게 있습니다.
③ 회원은 당 사이트 및 제 3자의 지적 재산권을 침해해서는 안 됩니다.
제 4 장 서비스의 이용
제 12 조 (서비스 이용 시간)
① 서비스 이용은 당 사이트의 업무상 또는 기술상 특별한 지장이 없는 한 연중무휴, 1일 24시간 운영을
원칙으로 합니다. 단, 당 사이트는 시스템 정기점검, 증설 및 교체를 위해 당 사이트가 정한 날이나 시간에
서비스를 일시 중단할 수 있으며, 예정되어 있는 작업으로 인한 서비스 일시중단은 당 사이트 홈페이지를
통해 사전에 공지합니다.
② 당 사이트는 서비스를 특정범위로 분할하여 각 범위별로 이용가능시간을 별도로 지정할 수 있습니다. 다만
이 경우 그 내용을 공지합니다.
제 13 조 (홈페이지 저작권)
① NDSL에서 제공하는 모든 저작물의 저작권은 원저작자에게 있으며, KISTI는 복제/배포/전송권을 확보하고
있습니다.
② NDSL에서 제공하는 콘텐츠를 상업적 및 기타 영리목적으로 복제/배포/전송할 경우 사전에 KISTI의 허락을
받아야 합니다.
③ NDSL에서 제공하는 콘텐츠를 보도, 비평, 교육, 연구 등을 위하여 정당한 범위 안에서 공정한 관행에
합치되게 인용할 수 있습니다.
④ NDSL에서 제공하는 콘텐츠를 무단 복제, 전송, 배포 기타 저작권법에 위반되는 방법으로 이용할 경우
저작권법 제136조에 따라 5년 이하의 징역 또는 5천만 원 이하의 벌금에 처해질 수 있습니다.
제 14 조 (유료서비스)
① 당 사이트 및 협력기관이 정한 유료서비스(원문복사 등)는 별도로 정해진 바에 따르며, 변경사항은 시행 전에
당 사이트 홈페이지를 통하여 회원에게 공지합니다.
② 유료서비스를 이용하려는 회원은 정해진 요금체계에 따라 요금을 납부해야 합니다.
제 5 장 계약 해지 및 이용 제한
제 15 조 (계약 해지)
회원이 이용계약을 해지하고자 하는 때에는 [가입해지] 메뉴를 이용해 직접 해지해야 합니다.
제 16 조 (서비스 이용제한)
① 당 사이트는 회원이 서비스 이용내용에 있어서 본 약관 제 11조 내용을 위반하거나, 다음 각 호에 해당하는
경우 서비스 이용을 제한할 수 있습니다.
- 2년 이상 서비스를 이용한 적이 없는 경우
- 기타 정상적인 서비스 운영에 방해가 될 경우
② 상기 이용제한 규정에 따라 서비스를 이용하는 회원에게 서비스 이용에 대하여 별도 공지 없이 서비스 이용의
일시정지, 이용계약 해지 할 수 있습니다.
제 17 조 (전자우편주소 수집 금지)
회원은 전자우편주소 추출기 등을 이용하여 전자우편주소를 수집 또는 제3자에게 제공할 수 없습니다.
제 6 장 손해배상 및 기타사항
제 18 조 (손해배상)
당 사이트는 무료로 제공되는 서비스와 관련하여 회원에게 어떠한 손해가 발생하더라도 당 사이트가 고의 또는 과실로 인한 손해발생을 제외하고는 이에 대하여 책임을 부담하지 아니합니다.
제 19 조 (관할 법원)
서비스 이용으로 발생한 분쟁에 대해 소송이 제기되는 경우 민사 소송법상의 관할 법원에 제기합니다.
[부 칙]
1. (시행일) 이 약관은 2016년 9월 5일부터 적용되며, 종전 약관은 본 약관으로 대체되며, 개정된 약관의 적용일 이전 가입자도 개정된 약관의 적용을 받습니다.