DOI: 10.25338/b8164c
title: Elucidating the origins of phycocyanobilin biosynthesis and phycobiliproteins

description: Terrestrial ecosystems and human societies depend on oxygenic
 photosynthesis, which began to reshape our atmosphere approximately 2.5
 billion years ago. The earliest known organisms carrying out oxygenic
 photosynthesis are the cyanobacteria, which use large complexes of
 phycobiliproteins as light-harvesting antennae. Phycobiliproteins rely on
 phycocyanobilin (PCB), a linear tetrapyrrole (bilin) chromophore, as the
 light-harvesting pigment that transfers absorbed light energy from
 phycobilisomes to the chlorophyll-based photosynthetic apparatus.
 Cyanobacteria synthesize PCB from heme in two steps: A heme oxygenase
 converts heme into biliverdin IXα (BV), and the ferredoxin-dependent bilin
 reductase (FDBR) PcyA then converts BV into PCB. In the current work, we
 examine the origins of this pathway. We demonstrate that PcyA evolved from
 pre-PcyA proteins found in nonphotosynthetic bacteria and that pre-PcyA
 enzymes are active FDBRs that do not yield PCB. Pre-PcyA genes are
 associated with two gene clusters. Both clusters encode bilin-binding
 globin proteins, phycobiliprotein paralogs that we designate as BBAGs
 (bilin biosynthesis-associated globins). Some cyanobacteria also contain
 one such gene cluster, including a BBAG, two V4R proteins, and an
 iron–sulfur protein. Phylogenetic analysis shows that this cluster is
 descended from those associated with pre-PcyA proteins and that
 light-harvesting phycobiliproteins are also descended from BBAGs found in
 other bacteria. We propose that PcyA and phycobiliproteins originated in
 heterotrophic, nonphotosynthetic bacteria and were subsequently acquired
 by cyanobacteria.
description: Absorption spectra were collected on Cary 50 or Cary 60
 spectrophotometers and were exported from the Cary software in .csv
 format. They were then processed using an in-house script to remove
 metadata and were plotted, analyzed, and prepared for presentation in
 Kaleidagraph. Presented spectra were exported from Kaleidagraph as
 tab-delimited text files with unix newlines and are deposited in that
 format. Some spectra were subtracted from each other to generate
 difference spectra and/or normalized to facilitate comparison; such
 spectra are presented separately in the same format. Some samples were
 characterized using a standard acid denaturation assay, and peak
 wavelengths from these spectra were plotted for comparison to references
 with known bilin composition. The reference and unknown values are
 deposited together as tab-delimited text files for short and long
 wavelengths. Circular dichroism spectra were collected
 on an Applied Photophysics Chirascan and were converted to tab-delimited
 text using the Applied Photophysics software. They were then plotted,
 analyzed, and prepared for presentation in Kaleidagraph. Presented spectra
 were exported from Kaleidagraph as tab-delimited text files with unix
 newlines and are deposited in that format. Fluorescence
 spectra were collected on a QM-6/2005SE fluorimeter equipped with
 red-enhanced photomultiplier tubes (Photon Technology International 814
 Series). Files were displayed in tabular format and copy/pasted for text
 export. They were then plotted, analyzed, and prepared for presentation in
 Kaleidagraph. Emission spectra were numerically integrated for estimation
 of fluorescence quantum yield, and the data used for quantum yield
 estimation are included as tab-delimited text along with files used for
 figure presentation (also as tab-delimited text).
 Deposited phylogenetic data include the three files used in the
 workflow for the published phylogenies. For each analysis, a multiple
 sequence alignment was constructed using MAFFT v7.450 (command-line
 settings --genafpair --maxiterate 16 --clustalout –reorder), deposited
 here in CLUSTAL format (.aln extension). The file was then converted to
 PHYLIP format with removal of gap-enriched columns (≥5%) using an in-house
 script. The PHYLIP format file is deposited here (.phy extension) and was
 used to infer a maximum-likelihood phylogeny in PhyML-3.1 with 100
 bootstraps (command-line settings -m WAG -d aa -s SPR -a e -c 4 -v e -o
 tlr -b 100). Statistical robustness was assessed using the transfer
 bootstrap expectation (TBE) as calculated in booster (software available
 at https://github.com/evolbioinfo/booster and file deposited in Newick format with .TBE.txt extension). For figure preparation, the Newick file output by Booster was processed in FigTree prior to annotation, scaling, and coloring in Adobe Illustrator. For each analysis, three files are deposited, with file extensions listed above. Homology models are presented for 3 proteins (POZ53557, CAP_1520, and MBL9008304) characterized in the course of this work. Models were generated starting from the same sequence alignment used for phylogenetic analysis of FDBRs, prior to removal of gap-enriched columns. Target and template sequences were filtered from this alignment using the alnfilter utility (available as part of the homolmapper distribution) and were converted to PIR format using the -convert option in CLUSTALW2. The .pir file was then manually edited to generate an input for MODELLER v9.22, and single homology models were generated for each target. These three models are deposited in PDB format. Synthetic gene sequences and the complete sequence for plasmid Spam-545/alt were generated using ApE and are deposited in flat text.
description: All files are reported as flat text files with unix
 newlines. A README.txt is included with this submission.