Photosynthesis Research

, Volume 141, Issue 3, pp 259–271 | Cite as

Cellular localization of tolyporphins, unusual tetrapyrroles, in a microbial photosynthetic community determined using hyperspectral confocal fluorescence microscopy

  • Meghan Barnhart-Dailey
  • Yunlong Zhang
  • Ran Zhang
  • Stephen M. Anthony
  • Jesse S. Aaron
  • Eric S. Miller
  • Jonathan S. LindseyEmail author
  • Jerilyn A. TimlinEmail author
Original Article


The cyanobacterial culture HT-58-2, composed of a filamentous cyanobacterium and accompanying community bacteria, produces chlorophyll a as well as the tetrapyrrole macrocycles known as tolyporphins. Almost all known tolyporphins (A–M except K) contain a dioxobacteriochlorin chromophore and exhibit an absorption spectrum somewhat similar to that of chlorophyll a. Here, hyperspectral confocal fluorescence microscopy was employed to noninvasively probe the locale of tolyporphins within live cells under various growth conditions (media, illumination, culture age). Cultures grown in nitrate-depleted media (BG-110 vs. nitrate-rich, BG-11) are known to increase the production of tolyporphins by orders of magnitude (rivaling that of chlorophyll a) over a period of 30–45 days. Multivariate curve resolution (MCR) was applied to an image set containing images from each condition to obtain pure component spectra of the endogenous pigments. The relative abundances of these components were then calculated for individual pixels in each image in the entire set, and 3D-volume renderings were obtained. At 30 days in media with or without nitrate, the chlorophyll a and phycobilisomes (combined phycocyanin and phycobilin components) co-localize in the filament outer cytoplasmic region. Tolyporphins localize in a distinct peripheral pattern in cells grown in BG-110 versus a diffuse pattern (mimicking the chlorophyll a localization) upon growth in BG-11. In BG-110, distinct puncta of tolyporphins were commonly found at the septa between cells and at the end of filaments. This work quantifies the relative abundance and envelope localization of tolyporphins in single cells, and illustrates the ability to identify novel tetrapyrroles in the presence of chlorophyll a in a photosynthetic microorganism within a non-axenic culture.


Bacteriochlorin Chlorophyll Cyanobacteria Membrane Multivariate curve resolution 



Charge-coupled device


Chlorophyll a


Classical least-squares


Hyperspectral confocal fluorescence microscopy


Multivariate curve resolution




Principal components analysis


Photodynamic inactivation


Scanning electron microscopy



The authors are grateful to Dr. Michael Sinclair for the use and maintenance of the hyperspectral confocal fluorescence microscope, and Howland Jones, Mark Van Benthem, David Melgaard, Mike Keenan, and David Haaland for original development of the MCR algorithm and software. We thank Dr. Philip Williams (University of Hawaii) for a gift of the HT-58-2 culture and a sample of tolyporphin A. This work was partially funded by the Photosynthetic Antenna Research Center (PARC), an Energy Frontier Research Center funded by the U.S. Department of Energy, Office of Science, Basic Energy Sciences under Award # DE-SC0001035 (to JAT, MBD and JSL). The NC State University Research and Innovation Seed Funding program also provided partial funding (to ESM and JSL). The Advanced Imaging Center at the Janelia Research Campus is a facility jointly supported by the Gordon and Betty Moore Foundation and the Howard Hughes Medical Institute (JSA). Sandia National Laboratories is a multimission laboratory managed and operated by National Technology and Engineering Solutions of Sandia LLC, a wholly owned subsidiary of Honeywell International Inc., for the U.S. Department of Energy’s National Nuclear Security Administration under contract DE-NA0003525. This paper describes objective technical results and analysis. Any subjective views or opinions that might be expressed in the paper do not necessarily represent the views of the U.S. Department of Energy or the United States Government.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflicts of interests.

Supplementary material

11120_2019_625_MOESM1_ESM.pdf (6.4 mb)
Supplementary material 1 (PDF 6504 KB)


  1. Bro R, DeJong S (1997) A fast non-negativity-constrained least squares algorithm. J Chemom 11 (5):393–401CrossRefGoogle Scholar
  2. Bruckner C (2017) Tolyporphin-an unusual green chlorin-like dioxobacteriochlorin. Photochem Photobiol 93(5):1320–1325. CrossRefPubMedGoogle Scholar
  3. Brzezowski P, Richter AS, Brim G (2015) Regulation and function of tetrapyrrole biosynthesis in plants and algae. Biochim Biophys Acta 1847:968–985CrossRefPubMedGoogle Scholar
  4. Collins AM, Liberton M, Garcia OF, Jones HDT, Pakrasi HB, Timlin JA (2012) Photosynthetic pigment localization and thylakoid membrane morphology are altered in Synechocystis 6803 phycobilisome mutants. Plant Physiol 158(4):1600–1609. CrossRefPubMedPubMedCentralGoogle Scholar
  5. De Juan A, Tauler R (2016) Multivariate curve resolution-alternating least squares for spectroscopic data. In: Ruckebusch C (ed) Resolving spectral mixtures, vol 30. Data handling in science and technology. Elsevier, Amsterdam, pp 5–52. CrossRefGoogle Scholar
  6. Haaland DM, Jones HDT, Timlin JA (2016) Experimental and data analytical approaches to automating multivariate curve resolution in the analysis of hyperspectral images. In: Ruckebusch C (ed) Resolving spectral mixtures, vol 30. Data handling in science and technology. Elsevier, Amsterdam, pp 381–406Google Scholar
  7. Haralick RM (1986) Statistical image texture analysis. In: Handbook of pattern recognition and image processing, vol 96. Academic Press, New York, pp 247–279Google Scholar
  8. Hood D, Niedzwiedzki DM, Zhang R, Zhang Y, Dai J, Miller ES, Bocian DF, Williams PG, Lindsey JS, Holten D (2017) Photophysical characterization of the naturally occurring dioxobacteriochlorin tolyporphin A and synthetic oxobacteriochlorin analogues. Photochem Photobiol 93(5):1204–1215. CrossRefPubMedGoogle Scholar
  9. Hughes RA, Zhang Y, Zhang R, Williams PG, Lindsey JS, Miller ES (2017) Genome sequence and composition of a tolyporphin-producing cyanobacterium-microbial community. Appl Environ Microbiol. CrossRefPubMedPubMedCentralGoogle Scholar
  10. Hughes RA, Jin X, Zhang Y, Zhang R, Tran S, Williams PG, Lindsey JS, Miller ES (2018) Genome sequence, metabolic properties and cyanobacterial attachment of Porphyrobacter sp. HT-58-2 isolated from a filamentous cyanobacterium–microbial consortium. Microbiology 164:1229–1239. CrossRefPubMedGoogle Scholar
  11. Jones HDT, Haaland DM, Sinclair MB, Melgaard DK, Collins AM, Timlin JA (2012) Preprocessing strategies to improve mcr analyses of hyperspectral images. J Chemom Intell Lab Syst 117:149–158. CrossRefGoogle Scholar
  12. Kato H, Komagoe K, Inoue T, Masuda K, Katsu T (2018) Structure-activity relationship of porphyrin-induced photoinactivation with membrane function in bacteria and erythrocytes. Photochem Photobiol 17:954–963. CrossRefGoogle Scholar
  13. Majumder EL, Wolf BM, Liu H, Berg RH, Timlin JA, Chen M, Blankenship RE (2017) Subcellular pigment distribution is altered under far-red light acclimation in cyanobacteria that contain chlorophyll f. Photosynth Res 134(2):183–192. CrossRefPubMedGoogle Scholar
  14. Martinez De Pinillos Bayona A, Mroz P, Thunshelle C, Hamblin M (2017) Design features for optimization of tetrapyrrole macrocycles as antimicrobial and anticancer photosensitizers. Chem Biol Drug Des 89:192–206. CrossRefGoogle Scholar
  15. Minehan TG, Cook-Blumberg L, Kishi Y, Prinsep MR, Moore RE (1999) Revised structure of tolyporphin A. Angew Chem Int Ed Engl 38(7):926–928CrossRefPubMedGoogle Scholar
  16. Morlière P, Maziere JC, Santus R, Smith CD, Prinsep MR, Stobbe CC, Fenning MC, Golberg JL, Chapman JD (1998) Tolyporphin: a natural product from cyanobacteria with potent photosensitizing activity against tumor cells in vitro and in vivo. Can Res 58(16):3571–3578Google Scholar
  17. Murton J, Nagarajan A, Nguyen AY, Liberton M, Hancock HA, Pakrasi HB, Timlin JA (2017) Population-level coordination of pigment response in individual cyanobacterial cells under altered nitrogen levels. Photosynth Res 134(2):165–174. CrossRefPubMedPubMedCentralGoogle Scholar
  18. Patterson GM, Baldwin CL, Bolis CM, Caplan FR, Karuso H, Larsen LK, Levine IA, Moore RE, Nelson CS, Tschappat KD (1991) Antineoplastic activity of cultured blue-green algae (cyanophyta). J Phycol 27(4):530–536. CrossRefGoogle Scholar
  19. Prinsep MR, Puddick J (2011) Laser desorption ionisation-time of flight mass spectrometry of the tolyporphins, bioactive metabolites from the cyanobacterium Tolypothrix nodosa. Phytochem Anal 22(4):285–290. CrossRefPubMedGoogle Scholar
  20. Prinsep MR, Caplan FR, Moore RE, Patterson GML, Smith CD (1992) Tolyporphin, a novel multidrug resistance reversing agent from the blue-green-alga Tolypothrix nodosa. J Am Chem Soc 114(1):385–387. CrossRefGoogle Scholar
  21. Prinsep MR, Patterson GML, Larsen LK, Smith CD (1995) Further tolyporphins from the blue-green-alga Tolypothrix nodosa. Tetrahedron 51(38):10523–10530. CrossRefGoogle Scholar
  22. Rippka R, Deruelles J, Waterbury JB, Herdman M, Stanier RY (1979) Generic assignments, strain histories and properties of pure cultures of cyanobacteria. Microbiology 111(1):1–61. CrossRefGoogle Scholar
  23. Saier MH Jr (2016) Transport protein evolution deduced from analysis of sequence, topology and structure. Curr Opin Struct Biol 38:9–17. CrossRefPubMedPubMedCentralGoogle Scholar
  24. Sinclair MB, Haaland DM, Timlin JA, Jones HDT (2006) Hyperspectral confocal microscope. Appl Opt 45(24):3283–3291. CrossRefGoogle Scholar
  25. Smith CD, Prinsep MR, Caplan FR, Moore RE, Patterson GM (1994) Reversal of multiple drug resistance by tolyporphin, a novel cyanobacterial natural product. Oncol Res 6(4–5):211–218PubMedGoogle Scholar
  26. Soares AR, Thanaiah Y, Taniguchi M, Lindsey JS (2013) Aqueous–membrane partitioning of β-substituted porphyrins encompassing diverse polarity. New J Chem 37(4):1087–1097. CrossRefGoogle Scholar
  27. Taniguchi M, Lindsey JS (2018) Database of absorption and fluorescence spectra of > 300 common compounds for use in PhotochemCAD. Photochem Photobiol 94(2):290–327. CrossRefPubMedGoogle Scholar
  28. Taniguchi M, Ptaszek M, Chandrashaker V, Lindsey JS (2017) The porphobilinogen conundrum in prebiotic routes to tetrapyrrole macrocycles. Orig Life Evol Biosph 47(1):93–119. CrossRefPubMedGoogle Scholar
  29. Tuceryan M, Jain AK (1993) Texture analysis. In: Chen CH, Pau LF, Wang PSP (eds) Handbook of pattern recognition and computer vision. pp 235–276.
  30. Vermaas WFJ, Timlin JA, Jones HDT, Sinclair MB, Nieman LT, Hamad S, Melgaard DL, Haaland DM (2008) In vivo hyperspectral confocal fluorescence imaging to determine pigment localization and distribution in cyanobacterial cells. Proc Natl Acad Sci 105(10):4050–4055. CrossRefPubMedGoogle Scholar
  31. Wang W, Kishi Y (1999) Synthesis and structure of tolyporphin A O,O-diacetate. Org Lett 1(7):1129–1132. CrossRefPubMedGoogle Scholar
  32. Zhang S, Lindsey JS (2017) Construction of the bacteriochlorin macrocycle with concomitant Nazarov cyclization to form the annulated isocyclic ring: analogues of bacteriochlorophyll a. J Org Chem 82(5):2489–2504. CrossRefPubMedGoogle Scholar
  33. Zhang Y, Zhang R, Nazari M, Bagley MC, Miller ES, Williams PG, Muddiman DC, Lindsey JS (2017) Mass spectrometric detection of chlorophyll a and the tetrapyrrole secondary metabolite tolyporphin A in the filamentous cyanobacterium HT-58-2. Approaches to high-throughput screening of intact cyanobacteria. J Porphyrins Phthalocyanines 21(11):759–768. CrossRefGoogle Scholar
  34. Zhang Y, Zhang R, Hughes RA, Dai J, Gurr JR, Williams PG, Miller ES, Lindsey JS (2018) Quantitation of tolyporphins, diverse tetrapyrrole secondary metabolites with chlorophyll-like absorption, from a filamentous cyanobacterium-microbial community. Phytochem Anal 29(2):205–216. CrossRefPubMedGoogle Scholar

Copyright information

© Springer Nature B.V. 2019

Authors and Affiliations

  1. 1.Bioenergy and Defense TechnologiesSandia National LaboratoriesAlbuquerqueUSA
  2. 2.Department of ChemistryNorth Carolina State UniversityRaleighUSA
  3. 3.Advanced Imaging Center, Janelia Research CampusHoward Hughes Medical InstituteAshburnUSA
  4. 4.Department of Plant and Microbial BiologyNorth Carolina State UniversityRaleighUSA

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