Effect of Producing Different Phenazines on Bacterial Fitness and Biological Control in Pseudomonas chlororaphis 30-84

유준명,Dongping Wang,Leland S. Pierson III,Elizabeth A. Pierson 한국식물병리학회 2018 Plant Pathology Journal Vol.34 No.1

Pseudomonas chlororaphis 30-84 is a biological control agent selected for its ability to suppress diseases caused by fungal pathogens. P. chlororaphis 30-84 produces three phenazines: phenazine-1-carboxylic acid (PCA), 2-hydroxy-phenazine-1-carboxylic acid (2OHPCA) and a small amount of 2-hydroxy-phenazine (2OHPHZ), and these are required for fungal pathogen inhibition and wheat rhizosphere competence. The two, 2-hydroxy derivatives are produced from PCA via the activity of a phenazine-modifying enzyme encoded by phzO. In addition to the seven biosynthetic genes responsible for the production of PCA, many other Pseudomonas strains possess one or more modifying genes, which encode enzymes that act independently or together to convert PCA into other phenazine derivatives. In order to understand the fitness effects of producing different phenazines, we constructed isogenic derivatives of P. chlororaphis 30-84 that differed only in the type of phenazines produced. Altering the type of phenazines produced by P. chlororaphis 30-84 enhanced the spectrum of fungal pathogens inhibited and altered the degree of take-all disease suppression. These strains also differed in their ability to promote extracellular DNA release, which may contribute to the observed differences in the amount of biofilm produced. All derivatives were equally important for survival over repeated plant/harvest cycles, indicating that the type of phenazines produced is less important for persistence in the wheat rhizosphere than whether or not cells produce phenazines. These findings provide a better understanding of the effects of different phenazines on functions important for biological control activity with implications for applications that rely on introduced or native phenazine producing populations.

Colletotrichum orbiculare에 대한 길항세균 Pseudomonas aurantiaca YC4963의 분리 동정 및 항균물질 Phenazine-1-carboxylic acid의 생산

채희정,김루미,문석식,안종웅,정영륜,Chae Hee-Jung,Kim Rumi,Moon Surk-Sik,Ahn Jong-Woong,Chung Young-Ryun 한국미생물학회 2004 미생물학회지 Vol.40 No.4

경남 지역의 근권 토양 및 식물뿌리 에서 다양한 길항세균을 분리하여 오이 탄저병원균 Colletotrichum orbiculare에 대한 길항효과를 조사하였다. 그중 국화과에 속하는 털진득찰 (Siegesbeckia pubescens Makino)뿌리에서 분리된 YC4963 균주가 병원균의 균사 생장에 대한 억제 능력이 가장 우수하였으며, 배양 상등액을 이용한 in vitro 실험에서도 C. orbiculare의 발아관 형성과 균사 생장을 억제하였다. 이 균은 Gram음성과 양성세균에 대 해서도 억제 능력이 있었으며, Botrytis cinerea, Fusalium oxysporum, Rhizoctonia solani 등의 다양한 식물병원균에 대해서도 억제 능력이 좋았다. 이 균주의 형태, 생리$\cdot$화학적 특성과 분자생물학적 특성을 조사한 결과 Pseudomonas aurantiaca로 동정되었다. 이 길항 세균이 분비하는 항생물질의 구조결정을 위하여 대량 배양 후 물질 분리와 여러 종류의 크로마토그래피를 수행하였다. 분리 정제하여 얻은 순수 물질은 노란색 바늘 모양의 결정체이었고, 질량 분석, FT-IR spectrum분석 및 NMP spectrum 분석을 바탕으로 구조를 추정한 결과 phenazine-1-carboxylic acid로 확인되었다. 이 항생물질의 활성을 조사하기 위하여 농도를 달리하여 C. orbiculare의 발아관 및 부착기 형성을 조사한 결과, 처리 18시간 후 발아관 생장은 103 ${\mu}m$로 대조구의 798 ${\mu}m$보다 8배 정도 억제되었으나 발아관과 부착기 형성비율은 큰 차이가 없었다. P. aurantiaca에 의한 phenazine-1-carboxylic acid의 생산과 이 항생물질에 의한 탄저병군의 억제효과는 본 연구에서 처음 보고되는 것이다.$생산 유전자가 전달되는 것을 확인하였다. Random amplified polymorphic DNA와 pulsed field gel electrophoresis분석을 사용하여 genomic DNA에 대한 유전형을 분석한 결과 균주간의 유전적 연관성은 매우 낮은 것으로 나타나 한 병원에서 발견되는 균주는 clonal spread에 의한 것이라는 일반적인 보고와 다른 결과를 얻었다., 체외순환을 시작한 이후부터는 2군에서 지속적으로 더 높은 경향이 있었으며(1군 $48.5\~64$ mL/min100 g, 2군 $65.8\~88.3$ mL/min/100 g), 특히 30분에서의 측정값은 통계적으로 유의한 차이를 보였다(1군$47.5{\pm}18.3\;mL/min100\;g,$ 2군$83.4{\pm}28.5\;mL/min100\;g,\;p=0.026$). 혈액 뇨질산, 크레아티닌, 그리고 혈장 용혈헤모글로빈의 변화는 두 군간에 차이가 없었다. 결론: 일정한 펌프 혈류 조건에서 박동성 혈류의 평균 혈압이 더 높다는 것은, 비박동성 혈류보다 조직관류압(Tissue Perfusion Pressure) 측면에서 우수하여 말초장기의 조직관류 효과에 유리한 요인이라고 볼 수 있다. 본 연구를 토대로 장시간의 체외순환에서는 신장기능을 대표하는 수치들에도 영향을 미칠 수 있으리라 예상되며, 신장 이외에 다른 주요 장기에 미치는 영향에 대한 연구를 더 진행할 필요가 있을 것으로 생각한다.예측 인자가 될 수 있을 것으로 생각된다.있을 것으로 판단된다.%$의 무기물질(Zeolite)이 첨가되어진 Modified California putting green system이 최적의 putting green 조건과 우수한 Bentgrass 잔디품질을 4년 동안 유지하였음을 이 실험을 통해 조사되어졌다. A bacterial strain YC4963 with antifungal activity against Colletotrichum orbiculare, a causal organism of cucumber anthracnose was isolated from the rhizosphere soil of Siegesbeckia pubescens Makino in Korea. Based on physiological and biochemical characteristics and 16S ribosomal DNA sequence analysis, the bac­terial strain was identified as Pseudomonas aurantiaca. The bacteria also inhibited mycelial growth of several plant fungal pathogens such as Botrytis cinerea, Fusarium oxysporum and Rhizoctonia solani on PDA and 0.1 TSA media. The antifungal activity was found from the culture filtrate of this isolate and the active compound was quantitatively bound to XAD adsorption resin. The antibiotic compound was purified and identified as phenazine-l-carboxylic acid on the basis of combined spectral and chemical analyses data. This is the first report on the production of phenazine-l-carboxylic acid by Pseudomonas aurantiaca.

페나진 유도체의 반응성과 항균성에 대한 양자 화학적 연구

한지훈(Ji-Hoon Han),이길준(Gil-Jun Lee) 대구과학대학교 국방안보연구소 2020 사회융합연구 Vol.4 No.5

그람 양성균과 그람 음성균에 대해 항균성을 가지며 7번 위치에 하이드록시기, 메톡시기, 메틸기, 아세틸기, 니트로기가 치환된 7-substituted 2,3-dihydroxy phenazine-5,10-dioxide 유도체들의 반응 부위의 예측과 항균성을 해석하기 위하여 ab initio 법으로 양자 화학적인 연구를 하였다. 최적화된 유도체들의 구조로부터 얻어진 frontier 오비탈과 frontier 에너지로부터 얻어진 결과는 유도체들은 친전자적인 분자였으며, 최적화된 페나진 고리 평면과 수직을 이루는 각 원자의 p오비탈이 주로 반응에 관여하고, 페나진 고리 속에 포함되어있는 질소 원자와 질소 원자와 결합한 산소가 친핵성 반응, 친전자성 반응, 친라디칼성 반응이 일어나는 주요 부위라는 결과를 얻었다. 그리고 5번 질소와 결합 된 산소의 친핵 frontier 밀도가 작을수록, 10번 질소와 결합한 산소의 친핵 frontier 밀도가 값이 클수록 유도체들이 가지는 항균력은 우세하였다. 연구의 결과는 새로운 페나진 유도체의 합성이나 효과적인 의약품의 설계에 유용한 정보들이다. A quantum chemical study for the reactivity site and antimicrobial activity of 7-substituted 2,3-dihydroxy phenazine-5,10-dioxide derivatives, which substituent are hydroxy, methoxy, methyl, acethyl and nitro group, known as antimicrobial agent, were investigated by ab initio method. The results obtained from the frontier orbital and frontier energy showed that the derivatives were electrophilic molecules, and the p orbitals of oxygen, nitrogen and carbon perpendicular to the optimized phenazine plane were mainly involved in the reaction. And it was found that the nitrogen atom in the phenazine ring and oxygen bonded to the nitrogen atom were the main sites where nucleophilic reactions, electrophilic reactions and radical reactions took place. As the nucleophilic frontier density of oxygen bound to nitrogen 5 was lower and it of oxygen bound to nitrogen 10 was higher, the antimicrobial activity of the derivatives was dominant. The results of this studies are useful information for the synthesis of new phenazine derivatives or the design of effective pharmaceuticals.

LasR Might Act as an Intermediate in Overproduction of Phenazines in the Absence of RpoS in Pseudomonas aeruginosa

( Qiuning He ),( Zhibin Feng ),( Yanhua Wang ),( Kewen Wang ),( Kailu Zhang ),( Le Kai ),( Xiuying Hao ),( Zhifen Yu ),( Lijuan Chen ),( Yihe Ge ) 한국미생물생명공학회(구 한국산업미생물학회) 2019 Journal of microbiology and biotechnology Vol.29 No.8

As an opportunistic bacterial pathogen, Pseudomonas aeruginosa PAO1 contains two phenazineproducing gene operons, phzA1B1C1D1E1F1G1 (phz1) and phzA2B2C2D2E2F2G2 (phz2), each of which is independently capable of encoding all enzymes for biosynthesizing phenazines, including phenazine-1-carboxylic acid and its derivatives. Other previous study reported that the RpoS-deficient mutant SS24 overproduced pyocyanin, a derivative of phenazine-1- carboxylic acid. However, it is not known how RpoS mediates the expression of two phz operons and regulates pyocyanin biosynthesis in detail. In this study, with deletion of the rpoS gene in the PAΔphz1 mutant and the PAΔphz2 mutant respectively, we demonstrated that RpoS exerted opposite regulatory roles on the expression of the phz1and phz2 operons. We also confirmed that the phz1 operon played a critical role and especially biosynthesized much more phenazines than the phz2 operon when the rpoS gene was knocked out in P. aeruginosa. By constructing the translational reporter fusion vector lasR’-’lacZ and the chromosomal fusion mutant PAΔlasR::lacZ, we verified that RpoS deficiency caused increased expression of lasR, a transcription regulator gene in a first quorum sensing system (las) that activates overexpression of the phz1 operon, suggesting that in the absence of RpoS, LasR might act as an intermediate in overproduction of phenazine biosynthesis mediated by the phz1 operon in P. aeruginosa.

Regulation of 3-Deoxy-D-arabinoheptulosonate-7-phosphate (DAHP) Synthase of Bacillus sp. B-6 Producing Phenazine-1-carboxylic acid

Kim, Kyoung-Ja Korean Society for Biochemistry and Molecular Biol 2001 Journal of biochemistry and molecular biology Vol.34 No.4

The 3-Deoxy-D-arabinoheptulosonate 7-phosphate (DAHP) synthase is the first enzyme of aromatic amino acid-, folic acid-, and phenazine-1-carboxylic acid biosynthetic pathways. DAHP synthase of Bacillus sp. B-6 that produces phenazine-1-carboxylic acid was feedback inhibited by two intermediary metabolites of aromatic amino acid biosynthetic pathways, prephenate and chorismate, but not by other metabolites, such as anthranilic acid, shikimic acid, p-aminobenzoic acid, and 3-hydroxyanthranilic acid. DAHP synthase of Bacillus sp. B-6 was not inhibited by end products, such as aromatic amino acids, folic acid, and phenazine-1-carboxylic acid. The inhibition of DAHP synthase by prephenate and chorismate was non-competitive with respect to erythrose 4-phosphate and phosphoenolpyruvate. Prephenate and chorismate inhibited 50% of the DAHP synthase activity at concentrations of $2{\times}10^{-5}\;M$ and $1.2{\times}10^{-4}\;M$, respectively The synthesis of DAHP synthase of Bacillus sp. B-6 was not repressed by exogenous aromatic amino acids, folic acid, and phenazine 1-carboxylic acid, single or in combinations.

Phenazine-1-carboxamide, an Extrolite Produced by Pseudomonas aeruginosa Strain (CGK-KS-1) Isolated from Ladakh and India, and its Evaluation Against Various Xanthomonas spp.

( K. Sirisha ),( C. Ganesh Kumar ),( Kallaganti Venkata Siva Ramakrishna ),( Shravan Kumar Gunda ) 한국미생물생명공학회(구 한국산업미생물학회) 2017 한국미생물·생명공학회지 Vol.45 No.3

In the enduring investigation of the bioactive microbes, Pseudomonas aeruginosa strain (referred to as CGK-KS-1 (ICTB-315)), isolated from Chumathang hot spring, Ladakh, and India, was identified to possess a major bioactive fraction with antimicrobial and anti-biofilm properties. This bioactive metabolite was purified through bioactivity-guided fractionation. The chemical structure of this major compound was elucidated as phenazine-1-carboxamide (PCN) based on ¹H and ¹³C NMR, FT-IR, EI-HR-MS and 2D NMR spectroscopic techniques. In the current study, PCN exhibited antimicrobial activity with MIC values ranging between 1.9-3.9 μg/ml against various test human pathogens and Xanthomonas spp. PCN showed the antibiofilm property with the IC₅₀ values ranging from 17.04 to 60.7 μM against different test pathogens. The in silico docking studies showed PCN strongly interacted with various proteins of different Xanthomonas spp. with high binding energies. We report herein for the first time the anti-biofilm property and the docking studies of PCN. The extrolite from P. aeruginosa strain CGK-KS-1 showed promising bioactivities and may be considered as a potential candidate for application in various biocontrol strategies.

vfr, A Global Regulatory Gene, is Required for Pyrrolnitrin but not for Phenazine-1-carboxylic Acid Biosynthesis in Pseudomonas chlororaphis G05

Wu, Xia,Chi, Xiaoyan,Wang, Yanhua,Zhang, Kailu,Kai, Le,He, Qiuning,Tang, Jinxiu,Wang, Kewen,Sun, Longshuo,Hao, Xiuying,Xie, Weihai,Ge, Yihe The Korean Society of Plant Pathology 2019 Plant Pathology Journal Vol.35 No.4

In our previous study, pyrrolnitrin produced in Pseudomonas chlororaphis G05 plays more critical role in suppression of mycelial growth of some fungal pathogens that cause plant diseases in agriculture. Although some regulators for pyrrolnitrin biosynthesis were identified, the pyrrolnitrin regulation pathway was not fully constructed. During our screening novel regulator candidates, we obtained a white conjugant G05W02 while transposon mutagenesis was carried out between a fusion mutant $G05{\Delta}phz{\Delta}prn::lacZ$ and E. coli S17-1 (pUT/mini-Tn5Kan). By cloning and sequencing of the transposon-flanking DNA fragment, we found that a vfr gene in the conjugant G05W02 was disrupted with mini-Tn5Kan. In one other previous study on P. fluorescens, however, it was reported that the deletion of the vfr caused increased production of pyrrolnitrin and other antifungal metabolites. To confirm its regulatory function, we constructed the vfr-knockout mutant $G05{\Delta}vfr$ and $G05{\Delta}phz{\Delta}prn::lacZ{\Delta}vfr$. By quantifying ${\beta}-galactosidase$ activities, we found that deletion of the vfr decreased the prn operon expression dramatically. Meanwhile, by quantifying pyrrolnitrin production in the mutant $G05{\Delta}vfr$, we found that deficiency of the Vfr caused decreased pyrrolnitrin production. However, production of phenazine-1-carboxylic acid was same to that in the wild-type strain G05. Taken together, Vfr is required for pyrrolnitrin but not for phenazine-1-carboxylic acid biosynthesis in P. chlororaphis G05.

vfr, A Global Regulatory Gene, is Required for Pyrrolnitrin but not for Phenazine-1-carboxylic Acid Biosynthesis in Pseudomonas chlororaphis G05

Xia Wu,Xiaoyan Chi,Yanhua Wang,Kailu Zhang,Le Kai,Qiuning He,Jinxiu Tang,Kewen Wang,Longshuo Sun,Xiuying Hao,Weihai Xie,Yihe Ge 한국식물병리학회 2019 Plant Pathology Journal Vol.35 No.4

In our previous study, pyrrolnitrin produced in Pseudomonas chlororaphis G05 plays more critical role in suppression of mycelial growth of some fungal pathogens that cause plant diseases in agriculture. Although some regulators for pyrrolnitrin biosynthesis were identified, the pyrrolnitrin regulation pathway was not fully constructed. During our screening novel regulator candidates, we obtained a white conjugant G05W02 while transposon mutagenesis was carried out between a fusion mutant G05ΔphzΔprn::lacZ and E. coli S17- 1 (pUT/mini-Tn5Kan). By cloning and sequencing of the transposon-flanking DNA fragment, we found that a vfr gene in the conjugant G05W02 was disrupted with mini-Tn5Kan. In one other previous study on P. fluorescens, however, it was reported that the deletion of the vfr caused increased production of pyrrolnitrin and other antifungal metabolites. To confirm its regulatory function, we constructed the vfr-knockout mutant G05Δvfr and G05ΔphzΔprn::lacZΔvfr. By quantifying β-galactosidase activities, we found that deletion of the vfr decreased the prn operon expression dramatically. Meanwhile, by quantifying pyrrolnitrin production in the mutant G05Δvfr, we found that deficiency of the Vfr caused decreased pyrrolnitrin production. However, production of phenazine-1-carboxylic acid was same to that in the wild-type strain G05. Taken together, Vfr is required for pyrrolnitrin but not for phenazine-1-carboxylic acid biosynthesis in P. chlororaphis G05.

Synthesis and cytotoxicity evaluation of substituted pyridazino[4,5-b] phenazine-5,12-diones and tri/tetra-azabenzofluorene-5,6-diones

Lee, Hyun-Jung,Kim, Jin-Sung,Suh, Myung-Eun,Park, Hyen Joo,Lee, Sang Kook,Rhee, Hee-Kyung,Kim, Hwa Jung,Seo, Eun-Kyung,Kim, Choonmi,Lee, Chong-Ock,Choo Park, Hea-Young 이화여자대학교 약학연구소 2008 藥學硏究論文集 Vol.- No.18

The substituted pyridazino[4,5-b]phenazine-5,12-diones and tri/tetra-azabenzo[a]fluorene-5,6-diones were synthesized from 6,7-dichloroph-thalazine-5,8-dione and 6,7-dichloroquinoline-5,8-dione, respectively. The cytotoxic activities of the prepared compounds were evaluated by an SRB (Sulforhodamine B) assay against the following human cancer cell lines: A549 (lung), SK-0V-3 (ovarian), SK-MEL-2 (melanoma), XF 498 (CNS), and HCT 15 (colon). Almost all synthesized pyridazino[4,5-b]phenazine-5,12-diones (7a-j) presented higher cytotoxicity than that of doxorubicin (IC_(50) = 0.097-0.225 μM) against the cancer cell lines, In particular, the cytotoxicity of compounds 7f (R₁=Et) and 7h (R₁, R₂ = Me) against all human cancer cell lines examined was about 10 times higher than that of doxorubicin. However, the cytotoxicities of several synthesized azabenzo[a]fluorene-5,6-diones (12a, 12c, 12d, 12e, and 12g) against the cancer cell lines in vitro were comparable to those of doxorubicin.

Pseudomonas aeruginosa BCNU 1204의 항균활성과 활성 물질

신화진(Hwa Jin Shin),주우홍(Woo Hong Joo) 한국생명과학회 2019 생명과학회지 Vol.29 No.1

신규 항세균물질을 탐색하는 사전조사에서 몇몇 분리균주들이 그람양성 세균과 그람음성 세균 모두에 항균활성을 보이며, 심지어 methicillin내성 Staphylococcus aureus (MRSA)에도 항균활성을 나타내었다. 이들 균주 중에서 한 균주가 표현형과 계통분석을 이용하여 특히 16S 리보좀 RNA 유전자 염기서열에 기초하여 Pseudomonas aeruginosa로 동정되었다. BCNU 1204 균주의 항균물질은 King`s medium B (pH 7.0)에서 35℃의 온도 조건으로 4일 배양 후 가장 최대로 생산되었다. 항균물질을 각종 유기용매로 분획한 결과, P. aeruginosa BCNU 1204의 dichloromethane (DCM)분획과 ethylacetate (EA) 분획이 그람 양성 세균에 강력한 항균활성을 보였으며, 특히 ethylacetate (EA) 분획이 methicillin내성 Staphylococcus aureus (MRSA)에 대하여 강한 항균활성을 나타내었다. Recycling preparative LC와 preparative TLC 로 활성물질 하나(분획 5-2)를 분리하여 GC-MS 분석한 결과 phenazine 화합물에 속하는 phenazine-1-carboxylic acid 로 동정하였다. 그리고 MRSA 균주에 대한 최소저해농도(minimum inhibitory concentration, MIC)가 MRSA균주인 CCARM 3089, 3090, 3091 그리고 3095 균주에 대하여 각각 25 μg/ml, 50 μg/ml, ≥25 μg/ml 그리고 ≥50 μg/ml 임을 확인하였다. 그러므로 P. aeruginosa BCNU 1204 분리균주는 항 MRSA 항생물질을 개발하기 위한 잠재 가치가 높은 생물자원으로 기대되며, P. aeruginosa BCNU 1204 균주로부터 리더 화합물을 획득하기 위한 보다 많은 연구가 요구된다. Previous screening of novel antibacterial agents revealed that some bacterial isolates exhibited antibiotic activity against both gram-positive and gram-negative bacteria and that they showed antibacterial activity, even against methicillin-resistant Staphylococcus aureus (MRSA). Among these isolates, one bacterial strain, BCNU 1204, was identified as Pseudomonas aeruginosa using phenetic and phylogenetic analysis, based on 16S ribosomal RNA gene sequences. The maximum productivities of antimicrobial substances of BCNU 1204 were obtained after being cultured at 35°C and pH 7.0 for 4 d in King’s medium B (KMB). Dichloromethane (DCM) and ethylacetate (EA) extracts of P. aeruginosa BCNU 1204 exhibited strong antimicrobial activity, particularly against gram-positive bacteria. The EA extracts exhibited broad-spectrum activity against antibiotic resistant strains. Fraction 5-2, was obtained by recycling preparative liquid chromatography (LC) and preparative thin-layer chromatography (TLC) and was identified as phenazine-1-carboxylic acid belonging to phenazines using gas chromatography and mass spectrometry (GC/MS). Its minimum inhibitory concentration (MIC) values were 25 μg/ml, 50 μg/ml, ≥25 μg/ml, and ≥50 μg/ml for MRSA CCARM 3089, 3090, 3091, and 3095 strains, respectively. P. aeruginosa BCNU 1204 may be a potential resource for the development of anti-MRSA antibiotics. Additional research is required to identify the active substance from P. aeruginosa BCNU 1204.