大腸菌의 Global Regulation에서의 polyamine의 役割 : Roles of polyamine in global regulation of Escherichia coli

국문 초록 (Abstract)

Polyamines(PA)은 진핵세포뿐만 아니라 원핵세포의 정상적인 성장에 필요하며,전사,번역,그리고 RNA,DNA,nucleotidetriphosphate에 결합을 통한 신호전달과 같은 세포내 다양한 범위의 과정에 관여하는 것으로 보고되어져 있다.일반적인 세포 생리에 주요하게 관여하는 것과 같은 중요성에도 불구하고,Lee,Tabor,Tkacheako그리고 Igarashi에 의해 PA의 기능적인 작용 기작이나 특이적인 target유전자가 부분적으로 알려져 있으나 여전히 잘 알려져 있지 않다.
생체내에서의 PA의 생리적인 역할과 작용기작을 규명하기 위하여, 본 연구에서는 Pho-regulon과 RpoS-regulon에 미치는 spermidine의 영향을 조사하였다.그리고 PA 생합성 돌연변이 결핍 대장균에서 PA 첨가유무에 따른 Proteomic분석을 수행하였다.
외부에서 첨가한 spermidine은 세균 AP의 합성과 phoB::lacZ의 전사발현을 촉진하였다. phoB::lacZ전사발현에서의 spermidine의 역할을 phoB, phoU,그리고 cya돌연변이 균주에서 심화 조사하였다. phoB돌연변이를 도입하면 spermidine의존적인 phoB::lacZ전사발현이 phosphate excess와 limitation 조건하에서의 PA 결핍에 의한 기저 수준 정도로 사라졌다.반면에 phoU돌연변이를 도입하면 phosphateexcess와 limitation 두 조건 모두에서 spermidine의 첨가유무에 관계없이 phoB::lacZ의 전사발현이 유사한 정도로 발현하였다.spermidine의존적인 phoB::lacZ의 발현을 cya돌연변이 균주에서 cAMP (1mM)를 첨가하고 또는 첨가하지 않고 조사한 결과, cya돌연변이에 의하여 phosphate excess와 limitation 조건하에서의 spermidine 의존적인 phoB::lacZ의 전사발현이 사라졌다. 돌연변의 균주의 spermidine 의존적인 phoB::lacZ의 결여는 phosphate excess와 limitation 조건하에서 cAMP (1mM)를 첨가함으로써 회복되었다.시험관에서는 spermidine이 PhoR과 PhoB의 인산화를 다양한 다른 반응 조건에 따라 다양한 방식으로 촉진하거나 저해하였다. Tn10 mutagenesis를 통하여 phoB::lacZ발현의 조절이 결여된 네 가지 돌연변이 균주를 선별하였다.이 중 세 돌연변이 균주의 Tn10삽입위치를 Inverse PCR로 염기서열을 확인한 결과 rbsA, yfiQ,그리고 ykgE와 100% 일치하였다. rbsA는 ribose 수송계의 결합 단백질의 한 부분으로 알려져 있다. yfiQ는 가상적인 NAD(P)와 ATP가 결합하는 acyl-CoA 합성효소였으며, ykgE는 glpC와 glcF와 유사하였다.
PA 생합성 균주는 정체기에 구형의 형태를 보이나,PA 생합성 결핍 돌연변이 균주는 rod형태의 신장된 형태를 보였다.외부에서 돌연변이 균주에 spermidine를 첨가하면 정체기의 비정상적인 형태가 대조군인 PA 생합성 균주에서와 같이 작고 구형의 형태로 회복되었다. 세포내의 PA 생합성 결핍에 의한 형태적인 변화의 작용기작을 밝히기 위하여, rpoS유전자의 전사와 번역에 대한 spermidine의 영향을 조사하였고 rpoS돌연변이를 PA 생합성 결핍 균주에 도입하였다.그 결과 외부에서 spermidine를 첨가하면 rpoS유전자의 전사와 번역이 증진되었다.PA 생합성 결핍 돌연변이 균주에 rpoS돌연변이를 도입한 결과 spermidine첨가유무에 관계없이 예상치 못한 auxotrophy 현상을 보였다. 이러한 rpoS-Spe-균주의 auxotrophy현상은 tryptophan을 첨가함으로써 억제되었다.또한 auxotrophy 현상을 부분적으로 억제하는 세 genomicclone를 선택하여 염기서열을 규명한 결과, flhDC, ccmABC,그리고 cobUST와 100% 일치함을 알 수 있었다. flhDC는 호기적 및 혐기적 호흡의 구성요소를 조절하는 조절자로 알려져 있다. ccmABC는 cytochrome c의 maturation에 관여하는 것으로 알려져 있으며, cobUST는 cobalamin(coenzymeB12)의 합성에 관여하는 것으로 알려져 있다.
PA 생합성 결핍 돌연변이 대장균 균주를 glucose 최소배지에서 PA를 첨가 또는 첨가하지 않고 키운 후 proteome분석을 하였다.그 결과 총 317개의 단백질이 PA 의존적인 차등적 발현을 보였다:이들 중 194개의 단백질들이 외부에서 첨가한 PA에 의해 up-regulation 되었고, 100개의 단백질들이 down-regulation 되었다.PA에 의해 유도되는 여섯개의 단백질들과, PA의 의해 감소하는 하나의 단백질을 mass spectrometry에 의하여 규명하였다.전자의 단백질들은 putrescine에 의해 유도되는 단백질들과 (ZnuA,TrpA,TrpB,TalB,GalM),spermidine에 의해 유도되는 단백질들과 (TrpA,TrpB,ZnuA,GalM,TpiA),그리고 cadaverine에 의해 유도되는 단백질들 (TrpA,TrpB,ZnuA,GalM,TpiA)이 있었다.후자의 단백질은 세 종류의 PA에 의해 모두 감소하며 LeuD로 규명되었다.

다국어 초록 (Multilingual Abstract)

Polyamines (PA) are necessary for normal cell growth in prokaryotic as well as eukaryotic cells, and participate in a wide range of cellular processes such as the modulation of transcription, translation, and signal transduction through binding to RNA, DNA, and nucleotide triphosphates. In spite of their importance as main participants in general cellar physiology, the specific target genes or functional mechanisms of PA are partially known by Lee, Tabor, Tkacheako and Igarashi but are still enigmatic.
To identify physiological role(s) and action mechanism(s) of PA in vivo, we investigated the effect of PA on the Pho-regulon and RpoS regulon. And PA deficient mutant E. coli were subjected to proteomic analysis with or without supplement PAs.
Exogenous Spd stimulates the bacterial AP synthesis and the phoB::lacZ expression in a PA-deficient strain. The role of spermidine in the phoB::lacZ was further examined in the phoB, phoU, and cya mutant backgrounds. Introduction of a phoB mutation abolished the spermidine-dependent stimulation of the phoB::lacZ expression to the basal level of the PA deficiency under both Pi excess and limitation conditions, while phoU mutation caused similar level of the phoB::lacZ expression under both Pi excess and limitation conditions regardless of the Spd supplementation. Regulation of the spermidine-dependent phoB::lacZ expression was further examined in a cya null mutant (cya::Km) backgrounds with or without supplement of cAMP (1mM) and found that a cya mutation abolished the spermidine-dependent stimulation of the phoB::lacZ expression under both Pi excess and limitation conditions. The lack of the spermidine-dependent phoB::lacZ expression of the mutant strain was suppressed by addition of cAMP (1mM) under both Pi excess and limitation conditions. In vitro, spermidine stimulates and inhibits the phosphorylation of PhoR and PhoB in various manner to different reaction conditions. By Tn10 mutagenesis, the regulatory defect of four mutants was isolated. Tn10 insertion sites of three mutants were identified by sequencing of inverse PCR products that gave a sequence with 100% identity to rbsA, yfiQ and ykgE. rbsA is the part of the binding-protein-dependent transport system for ribose. yfiQ is the putative NAD(P)-binding and ATP binding acyl-CoA synthetase. ykgE is similar to glpC and glcF.
PA-proficient strains exhibit spherical morphology in stationary phase, wherase a PA-deficient mutant strain was found to remain rod shaped and elongated. Exogenous spermidine supplement to the mutant suppressed the abnormality of the morphology at the stationary-phase, becoming smaller and more spherical as in the isogenic PA-proficient control strain. As an effort to elucidate the mechanism of the morphological changes caused by the endogenous polyamine deficiency, we investigate the effect of PA on the rpoS transcriptional and translational expression and introduced a rpoS mutation to the PA-deficient background. Exogenous spermidine enhanced the rpoS transcriptional and translation expression. The rpoS in the polyamine-deficient background showed an unexpected auxotrophy regardless of the polyamine supplement. And auxotrophy of the rpoS-Spe- was suppressed by addition of tryptophan. Also, three genomic clones partially suppressing the auxotrophy were selected, and were identified by sequencing that gave a sequence with 100% identity to flhDC, ccmABC, and cobUST. flhDC is the regulator of aerobic and anaerobic respiratory components. ccmABC is involve in the cytochrome c maturation. cobUST is involved in cobalamin (coenzyme B12).
Cultures of a PA-deficient mutant E. coli grown in glucose minimal medium with or without supplement of PAs, were subjected to proteome analysis. Total of 317 proteins were positively identified to show PA-dependent differential expression: 194 were up-regulated, and 100 showed down-regulation by the exogenous PA addition. Six of the PA-inducible and one of the PA-repressible proteins were identified by mass spectrometry. The former proteins included putrescine-induced proteins (ZnuA, TrpA, TrpB, TalB, GalM), spermidine-induced proteins (TrpA, TrpB, ZnuA, GalM, TpiA), and cadaverine-induced proteins (TrpA, TrpB, GalM, ZnuA, TpiA). The latter protein, repressed by all of the three PAs tested, was identified as LeuD.

목차 (Table of Contents)

• CONTENTS
• List of Tables = ⅶ
• List of Figures = ⅷ
• Abbreviations = ⅹⅰ
• Ⅰ. INTRODUCTION = 1
• 1. PAs = 1
• 2. Global Regulation = 16
• Ⅱ. MATERIALS AND METHODS = 19
• 1. Strains, bacteriophages and plasmids = 19
• 2. Media and culture conditions = 19
• 3. DNA manipulations = 23
• 4. Genetic manipulations = 25
• 5. Assays = 32
• 6. Protein manipulations = 33
• 7. RNA manipulation = 37
• 8. Microphotograph = 37
• 9. Proteome analysis = 38
• Ⅲ. RESULTS = 41
• Ⅰ. Role of PAs in the regulation of Pi(Pho)-regulon in E.coli = 41
• 1. Bacterial AP activity is increased by Spd = 41
• 2. phoB::lacZ expression is increased by Spd at the basal and inducible level expression = 41
• 3. Spd concentration-dependent phoB::lacZ expression = 41
• 4. Spd-dependent phoB expression requires phoB = 44
• 5. cya affects the Spd-dependent phoB::lacZ expression = 44
• 6. Spd-dependent phoB::lacZ expression requires both Spd and cAMP = 48
• 7. Multicopy phoBR partially increased Spd-dependent phoB::lacZ expression in basal level expression = 48
• 8. Spd-dependent phoB::lacZ expression is reduced in anaerobic condition = 48
• 9. Spd affects the ATP concentration = 52
• 10. The Spd dependent bacterial AP activity and phoB::lacZ expression in a phoU mutant background = 55
• 11. A mutation derepressing Pho regulon partially suppressed the abnormal growth of PA-deficient mutant =55
• 12. Isolation and characterization of transposon-mediated mutations affecting PA-dependent phoB::lacZ expression = 58
• 13. Effect of Spd on the phosphorylation of PhoR and PhoB = 60
• 13.1. Effect of Spd on the phosphorylation of PhoR and PhoB in vivo = 60
• 14. Effect of Spd on the phosphorylation of PhoR and PhoB in vitro = 63
• Ⅱ. Stimulation of the stationary sigma factor, RpoS, of E. coli = 70
• 1. PA affects the morphology of stationary-phase E. coli = 70
• 2. rpoS transcriptional and translational expression is increaced by Spd = 70
• 3. rpoS transcriptional expression is changed in various starvation conditions = 74
• 4. Tryptophan auxotrophy caused by rpoS::Tn10 in the PA-deficient background 74
• Ⅲ. Proteomic analysis of proteins differentially regulated by PAs in E. coli = 83
• 1. 2-D gel analysis of cells grown in minimal medium with or without PAs = 83
• 2. Quantitative measurement of protein expression levels = 83
• 3. Identifying members of the Put, Spd and Cad stimulons = 84
• 4. PA and tryptophan requirement = 84
• Ⅳ. DISCUSSION = 102
• 1. Role of PAs in the regulation of Pi (Pho)-regulon in E. coli = 103
• 2. PA-dependent rpoS Expression = 105
• 3. PA-dependent Protein Expression = 107
• 4. Role of PA on the global regulation = 108
• Ⅵ. REFERENCES = 111
• Abstract = 124
• 국문초록 = 128