A Site Directed Mutagenesis Approach Reveals Natural Evolutionary Footprints of Cyanobacterial Phytochromes

다국어 초록 (Multilingual Abstract)

Cyanobacteria have evolved diverse bilin-bound photoreceptors, phytochromes, to regulate their numerous adaptive mechanisms in response to ever changing light information. In addition to canonical bacterial (cBphp), cyanobacterial (Cph1) type phytochromes (Phys), and dual Cys containing Tandem Cys Cyanobacterial phytochrome (TCCP), survey of cyanobacterial genomes publicly available exhibited additional diversity layer with respect to the number of bilin-linkage residue, cysteine (Cys) as well as their locations at photosensory core module. Namely, we screened two novel types of cyanobacterial phytochromes (Cphs), 1) with two Cys residues that are separately arranged at the N-terminal PAS domain and GAF domain (named hereafter separately arranged dual Cys cyanobacterial phytochrome, SDCP) and 2) triple Cys residues, one at the PAS domain and two at GAF domain (named hereafter triple-Cys Phy, TCP). These two Cphs, together with TCCP family, are mostly harbored by heterocyst-forming cyanobacteria. SDCP exhibits a typical red/far-red photocycle with heterogenous dark formed 15Z states with BV, whilst blue/orange photocycle was observed in TCP, consistent with TCCP photoconversion. They were all PCB-binders when expressed in unicellular cyanobacterium Synechocystis sp. PCC6803. Mutagenesis study revealed that both Cys residues in SDCPs were functional acting as bilin-linkage residues, whilst Cys residue in PAS of TCP was dispensable. In contrast to TCCP with pH dependent absorption changes in its 15E state, TCP exhibited protochromicity only in its 15Z state. By addition and deletion of Cys residue to PAS and/or GAF of other types of Cphs via site-directed mutagenesis, we demonstrated that a single type of Cph is artificially converted to other types of Cphs, suggesting that TCCP and TCP are the most recently diverged Phy families. Collectively, our studies concluded that acquisition of dual Cys located at GAF domain enable cyanobacteria to improve plasticity of their survival strategy spanning from red to blue band limit.

국문 초록 (Abstract)

남세균은 끊임없이 변화하는 빛 정보에 반응하여 수많은 적응 메커니즘을 조절하기 위해 다양한 빌린에 결합하는 광단백질중 하나인, 파이토크롬을 진화 시켰습니다. 공개적으로 사용 가능한 남세균들의 게놈 조사는 전형적인 박테리아 타입 (cBphp), 남세균 타입 (Cph1) 유형의 파이토크롬롬 (Phys) 및 Tandem Cys 즉, 이중 Cys를 가지는 남세균 타입 (TCCP)의 파이토크롬들 외에도 파이토크롬에서 빌린에 결합 할 수 있는 아미노산의 수 그리고 이 아미노산들이 광반응 모듈에서 어디에 위치해 있는지와 관련하여 더욱 다양한 파이토크롬이 존재한다는 것을 밝혀낼 수 있게 하였습니다. 저희는 이번에 대중에게 공개된 남세균 게놈들을 조사함으로써 2가지 새로운 유형의 남세균 파이토크롬을 발견했습니다. 첫 번째는 각각 파스와 가프 도메인에 빌린에 결합하는 Cys 잔기가 있는 SDCP (두개의 Cys잔기가 떨어져 있는 남세균 파이토크롬) 두 번째는 3개의 빌린에 결합하는 Cys잔기를 가지는 파이토크롬인 TCP 인데 TCP에서는 Cys가 하나는 파스 도메인에 있고 나머지 2개는 서로 tandem하게 나열되어 있는 형태로 가프 도메인에 위치합니다. TCCP와 함께 이 새롭게 발견된 두 종류의 파이토크롬은 대부분이 히테로시스트라고 불리는 질소를 고정하는 부위를 가지는 남세균의 게놈에서 발견이 됩니다. SDCP의 경우 전형적인 파이토크롬처럼 15Z 형태에서는 적색 파장의 빛과 15E 형태에서는 근적색 파장의 빛에 반응을 하지만 빌리버딘과 결합했을 때에는 다른 파이토크롬에서는 발견되지 않은 특이한 형태의 이질적인 15Z 형태를 가집니다. TCP의 경우 TCCP처럼 15Z 에서는 파란색 파장 15E 에서는 주황색 파장의 빛에 반응을 합니다. 이들을 남세균에서 발현 시켰을땐 모두 PCB에 결합을 합니다. 돌연변이 유발 접근방식을 취했을 때 SDCP의 경우 두 Cys 잔기가 모두 빌린에 결합을 하였고 TCP는 가프 도메인에 있는 Cys잔기만 빌린에 결합을 하였습니다. 15E 형태에서 PH에 반응을 했던 TCCP와 달리 TCP는 15Z 형태에서 PH에 반응을 하였습니다. 돌연변이 접근 방식을 다른 전형적인 남세균 파이토크롬에서도 적용을 시켜 Cys 잔기를 파스 도메인 및 가프 도메인에 첨가하거나 제거했을 때 한 종류의 파이토크롬이 다른 종류의 파이토크롬으로 인위적으로 바뀔 수 있다는 것을 증명했습니다. 그리고 TCP와 TCCP가 남세균 파이토크롬 중에서 가장 최근에 진화했다는 것도 저희 실험을 통해 제안할 수 있었습니다. 종합적으로 저희 실험을 통해서 남세균의 파이토크롬은 가프 도메인에 두 개의 tandem에 나열된 Cys 잔기를 얻고 적색빛에서 파란빛으로 흡수할 수 있는 파장 범위를 넓힘으로써 남세균들이 주어진 환경에 살아날을 수 있기 위한 유연성을 개선시켰다고 결론을 지을 수 있었습니다.

목차 (Table of Contents)

• LIST OF TABLES...........................................................................................................iii
• LIST OF FIGURES.........................................................................................................iV
• ABSTRACT......................................................................................................................Vi
• 1. INTRODUCTION..........................................................................................................1
• 2. MATERIALS AND METHODS..................................................................................4
• 3. RESULTS.....................................................................................................................20
• 3.1. Phylogenetic analysis and multiple sequence alignments revealed the novel type of phytochromes and cBphps, and their relationship with cyanobacterial evolution...................................................................................20
• 3.2. Dual-Cys cBphps (SDCP) establish R/Fr photocycle similar to Cph1 and cBphps, whilst triple-Cys phytochrome (TCP) exhibits VB/YO photoconversion similar to those of TCCP...................................................31
• 3.3. Both Cys residues in PAS and GAF domain of SDCPs form covalent linkage with chromophore, exhibiting red/far-red photocycle......................34
• 3.4. Cphs are color-tunable by insertion/deletion of Cys residue(s) located at respective PAS and GAF domains............................................................39
• 3.5. PCB is preferred chromophore in cyanobacteria....................................56
• 3.6. Artificial green and red photocycle tuning of Cphs is only observed in TCCP............................................................................................................59
• 3.7. Partial pH dependent spectral change observed in LeTCP...................61
• 3.8. Cryo-EM image revealed high similarity between FiSDCP and Agp1.....................................................................................................................64
• 4. DISCUSSION...............................................................................................................65
• 4.1. Heterogenous population of BV-SDCPs implies the presence of two thioether linkages to A-ring of BV.................................................................68
• 4.2. Site directed mutagenesis reveals TCP as the latest comer in Cph evolution................................................................................................71
• 4.3. Locking in dark state at low temperature is unique characteristics of SDCPs............................................................................................................74
• 5. REFERENCES.............................................................................................................76
• 국문초록...........................................................................................................................81
• LIST OF TABLES
• Table 1. Primers used for construct of overexpression strain of Synechocystis sp. PCC6803..............................................................................................................................5
• Table 2. Primers used for construct of in vitro expression in E.coli.........................8
• Table 3. Spectral properties of phytochromes and their variants used in
• this study...........................................................................................................................47
• LIST OF FIGURES
• Figure 1. Distritbtion of phytochromes (A) and their evolutionary relationship (B) in cyanobacteria................................................................................................................23
• Figure 2. Evolutionary relationship between cyanobacteria (right) and their
• phytochromes(left).............................................................................................................27
• Figure 3. Sequence alignment of PCMs derived from Cph1, Bphp, cBphp, SDCP, TCP, and TCCP type phytochromes..............................................................................30
• Figure 4. Full-length domain architectures of phytochromes used
• in this study......................................................................................................................33
• Figure 5. Photoconversion of SDCPs (A) and TCPs (B)
• with BV and PCB adducts.............................................................................................37
• Figure 6. Dark-minus-light difference spectra of BV and PCB bound phytochromes and their variants used in this study.............................................................................38
• Figure 7. Absorbance spectrum for BV and PCB bound PCM of wt and variants of DrBphp (A), cBphp (Tpr4568 and Tpl6224) (B), SyCph1, ToCph1 (C), and ToTCCP (D)......................................................................................................................42
• Figure 8. Dark-minus-light difference spectra of BV and PCB bound phytochromes and their variants used in this study.............................................................................44
• Figure 9. Denatured different spectra of BV and PCB bound phytochrome PCMs isolated from E.coli..........................................................................................................45
• Figure 10. Zinc-fluorescence and CBB staining image for PCMs of phytochomres
• isolated from E.coli..........................................................................................................46
• Figure 11. Photoconversion of recombinant Cphs isolated from the unicellular
• cyanobacterium Synechocysis sp. PCC 6803.................................................................57
• Figure 12. Dark-minus-light difference spectra of PCMs isolated from
• overexpression strains of Synechocystis sp. PCC6803.................................................57
• Figure 13. Zinc-fluorescence and CBB staining image for PCMs of phytochomres
• isolated from overexpression strains...............................................................................58
• Figure 14. Denatured different spectra of phytochrome PCMs isolated from
• overexpression strains.......................................................................................................58
• Figure 15. X-CS variants of TCPs with R/Fr photocycle..........................................60
• Figure 16. PH dependence of 15Z and 15E state of PCB bound SDCPs, BV
• bound SDCP, PCB bound LeTCP, and PCB bound NcSDCP variant (C-CC)........63
• Figure 17. PH shift experiment reveals partial PH dependency in 15Z state of
• PCB bound LeTCP...........................................................................................................63
• Figure 18. Cryo-EM image of PAS-GAF of FiSDCP................................................65
• Figure 19. Superimposed Phy and miRFP670 structures............................................66
• Figure 20. Proposed heterogenous dark state BV adducts in SDCPs.......................67
• Figure 21. Heterogenous dark state BV adducts in SDCPs.......................................70
• Figure 22. Scheme for Cph evolutionary pathway......................................................73