Responses of a Newly Evolved Auxotroph of Chlamydomonas to B₁₂ Deprivation

Abstract

One-sentence summary: Study of an artificially evolved strain of the model alga Chlamydomonas provides insight into how algal B 12 auxotrophy has arisen naturally but also reveals the challenges to becoming B 12 dependent.

Exploring the onset of B12-based mutualisms using a recently evolved Chlamydomonas auxotroph and B12-producing bacteria

“…The three strains were cultured for seven days with several B 12 concentrations. As previously demonstrated (Bunbury et al ., 2020), metE7 maximal cell density showed a clear dose-response to vitamin B 12 , while the growth of the ancestral and revertant strains (both containing a functional METE gene) was not increased by B 12 (Figure 1A, Figure S2). Similarly, the effect of B 12 supplementation on M. loti was quantified for both the wild-type strain and a B 12 synthesis mutant (bluB - ;(Shimoda et al ., 2008)), which was confirmed to be unable to synthesise B 12 (Figure S3B).…”

“…One potential explanation may be that the B 12 -limited metE7 cells released (including through cell death) a greater amount of organic carbon or a different spectrum of compounds leading to a greater bacteria:algal ratio. In fact, increased cell size, cell death and an increase in starch and triacylglycerides were all found to occur in metE7 on B 12 deprivation (Bunbury et al ., 2020). Figure 3A clearly indicates that the addition of glycerol allows a large increase in M. loti growth and subsequently B 12 production (Figure 3C), which in turn is the likely cause of the increase in metE7 cell density.…”

“…Nutrient amendment experiments of aquatic ecosystems have revealed that B 12 or B 12 -producers frequently limit phytoplankton growth (Bertrand et al ., 2007; Koch et al ., 2011; Paerl et al ., 2015; Joglar et al ., 2020; Barber-Lluch et al ., 2021), and laboratory experiments have investigated the effect of B 12 supply on phytoplankton physiology (Bertrand et al ., 2012; Heal et al ., 2019; Koch and Trimborn, 2019; Nef et al ., 2019). Studies have revealed the effects of B 12 on the methionine cycle, C1 metabolism, and cell growth and division in Euglena gracilis (Shehata and Kempner, 1978; Carell and Seeger, 1980), Tisochrysis lutea (Nef et al, 2019), Thalassiosira pseudonana (Heal et al, 2019) , and Chlamydomonas reinhardtii (Bunbury et al, 2020) . In these species, B 12 acts as a cofactor for the enzyme methionine synthase (METH), although some, like C. reinhardtii , also encode a B 12 -independent isoform (METE).…”

The Spermidine Synthase Gene SPD1: A Novel Auxotrophic Marker for Chlamydomonas reinhardtii Designed by Enhanced CRISPR/Cas9 Gene Editing

“…Earliest reports of auxotrophies date back to the 1950s [ 30 ], but as of today, only five auxotrophic selection systems are established in C. reinhardtii [ 1 , 14 , 28 ]: Arginine biosynthesis ( ARG9 , OTC1 , AGS1 , ARG7 ), the only amino acid auxotrophy, nitrate assimilation ( NIT1/2 ), phototrophic growth (acetate dependency; OEE1, ATPC ) and biosynthesis of the vitamins nicotinamide ( NIC7 ) and thiamine ( THI4/10 ). The gene responsible for a p -aminobenzoic acid auxotrophy [ 30 , 31 ] has not been reported yet, but a recently discovered cobalamin (B 12 , METE ) auxotrophy could be utilized in the future [ 32 , 33 ].…”

The Spermidine Synthase Gene SPD1: A Novel Auxotrophic Marker for Chlamydomonas Reinhardtii Designed by Enhanced CRISPR/Cas9 Gene Editing

“…Earliest reports of auxotrophies date back to the 1950s [30], but as of today, only five auxotrophic selection systems are established in C. reinhardtii [1,14,28]: Arginine biosynthesis (ARG9, OTC1, AGS1, ARG7), the only amino acid auxotrophy, nitrate assimilation (NIT1/2), phototrophic growth (acetate dependency; OEE1, ATPC) and biosynthesis of the vitamins nicotinamide (NIC7) and thiamine (THI4/10). The gene responsible for a p-aminobenzoic acid auxotrophy [30,31] has not been reported yet, but a recently discovered cobalamin (B12, METE) auxotrophy could be utilized in the future [32,33].…”