Abstract
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.
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Abbreviations
- CCD:
-
Charge-coupled device
- Chl:
-
Chlorophyll a
- CLS:
-
Classical least-squares
- HCFM:
-
Hyperspectral confocal fluorescence microscopy
- MCR:
-
Multivariate curve resolution
- PBS:
-
Phycobilisomes
- PCA:
-
Principal components analysis
- PDI:
-
Photodynamic inactivation
- SEM:
-
Scanning electron microscopy
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Acknowledgements
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.
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Barnhart-Dailey, M., Zhang, Y., Zhang, R. et al. Cellular localization of tolyporphins, unusual tetrapyrroles, in a microbial photosynthetic community determined using hyperspectral confocal fluorescence microscopy. Photosynth Res 141, 259–271 (2019). https://doi.org/10.1007/s11120-019-00625-w
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DOI: https://doi.org/10.1007/s11120-019-00625-w