Background and goals

Origin and characterization of the cpc1-1 mutant

The cpc1-1 mutant was initially generated through insertional mutagenesis using a nitrate reductase (NIT1)-selectable marker in a nitrate reductase-deficient host strain (nit1-305, CC-2677) [4]. The transformation approach resulted in a genomic insertion that disrupted the CPC1 gene, which encodes a ~350 kDa protein localized to the central pair apparatus of the flagellar axoneme. Electron microscopy revealed that the cpc1-1 mutation specifically disrupts the C1b projection of the central pair complex, leading to altered flagellar waveform and reduced swimming velocity [4].

Further biochemical analysis identified that CPC1 contains an adenylate kinase domain and interacts with enolase and other glycolytic enzymes, suggesting a potential role in local ATP regulation within the flagellar axoneme [5]. We recently reported that the motility defects in the cpc1-1 strain can be reversed with drugs that may impact metabolism [2], but CPC1's role in metabolic pathways hasn’t been explored.

Genetic background considerations

The cpc1-1 mutation was generated in strain CC-2677 (nit1-305), a genetic lineage distinct from the widely used 137c laboratory background strains (CC-124/CC-125). After initial characterization, the cpc1-1 mutation underwent backcrossing "at least four times" into the 137c background to generate the strain CC-3707 used for studies of central pair complex function [4][5]. While this recurrent backcrossing has likely incorporated the majority of the 137c genetic background (theoretically at least 94% after four backcrosses [6]), portions of the original CC-2677 genome will persist in the current CC-3707 strain. However, the 137c strain CC-124 remains the best wild-type control for cpc1-1 since the majority of the mutant’s genome is derived from the 137c lineage. The characteristic reduced flagellar beat frequency in this mutant was rigorously mapped to the CPC1 locus through comprehensive genetic approaches, including multiple independent alleles exhibiting complementation failure [4], so we can safely infer causality between CPC1 disruption and the observed motility defect.

The ida4 mutant (CC-2670) exhibits a defect in the inner dynein arm I-projection resulting in the loss of specific dynein subspecies from the axoneme and reduced swimming velocity. Unlike cpc1-1, this mutation was introduced in the 137c background without extensive backcrossing from a divergent strain [7][8].

Chlamydomonas smithii (CC-1373) is fully interfertile with C. reinhardtii but has a distinct genetic background with significant sequence polymorphisms [9]. Importantly, unlike standard laboratory strains of C. reinhardtii (CC-124/CC-125), which carry nit1 and nit2 mutations preventing growth on nitrate as a sole nitrogen source, C. smithii retains functional nitrate assimilation pathways [10][11][12]. This strain, therefore, serves as a positive control for the ability to utilize nitrate.

Finally, C. reinhardtii strain CC-5415 (nit1 agg1) is a cross between nit1-305 and CC-124 and we included it as an additional control because, like strain CC-3707, it contains genetic contributions from both nit1-305 and 137c backgrounds. However, due to the multiple generations of backcrossing, CC-3707 retains a substantially higher proportion of the 137c genome than CC-5415.

Goals

We wanted to understand whether ida4 and cpc1-1 mutants exhibit growth phenotypes distinct from wild-type strains across various media compositions, indicative of metabolic defects. We sought to:

1. Characterize growth patterns of cpc1-1 and ida4 compared to wild-type strains on standard and nutrient-enriched media
2. Assess nitrogen utilization capabilities across different nitrogen sources

The approach

We compared growth of wild-type Chlamydomonas smithii (CC-1373) and Chlamydomonas reinhardtii (CC-124, CC-5415) strains, the cpc1-1 mutant (CC-3707), and the ida4 mutant (CC-2670) across multiple media formulations to identify metabolic influences of these mutations. We controlled for initial cell density to ensure precise quantification of growth differences. All strains were transferred to these different media after growth under the same culturing conditions to avoid environmental influences.

Strains and culture conditions

We examined growth for the following Chlamydomonas strains (Table 1).

Additionally, we included the adenosine deaminase mutant strain CLIP2.048478 in the experiments. However, we didn't include these results in the text because we took cells directly from the Chlamydomonas Resource Center inoculum, not from a single colony we’d PCR-confirmed to have the mutant ada allele. Therefore, we can't claim that the ada mutant allele causes any potential growth phenotypes we observed. However, we include the data in a supplemental figure.

We maintained all strains on standard TAP (tris-acetate-phosphate) medium with 1.5% agar under 12:12 light:dark cycles at ambient temperature.

Experimental setup and cell preparation

We harvested cells from TAP + 1.5% agar plates and suspended them in approximately 1 mL of liquid TAP medium. We passed all suspensions through 5 μm filters via centrifugation to ensure uniform cell populations and eliminate aggregates.

We took initial optical density measurements at 730 nm using a SpectraMax iD3 plate reader (Molecular Devices) with samples loaded into a black-walled 96-well plate. We normalized cell suspensions to the lowest concentration (OD₇₃₀ = 0.043) by diluting with TAP medium according to calculated ratios. We then maintained the plate under static conditions overnight with a 12:12 light:dark cycle.

Serial dilution and plating

The next day, we prepared serial dilutions (1:1, 1:10, 1:100, 1:1000, 1:10,000) of the normalized cell suspensions by successively transferring 30 μL aliquots into wells containing 270 μL of fresh TAP medium. For each strain and dilution, we spotted 2 μL onto solid media plates in triplicate (except for soil extract medium, which had two replicates due to contamination issues).

Growth media formulations

We tested growth across multiple media formulations to assess strain-specific responses to different nutrient compositions (Table 2).

We prepared all media formulations with 1.5% agar and stored them at 4 °C until use. The age of the plates ranged from 4 to 10 months because we limited the experimental design to readily available materials. Before inoculation, we equilibrated the plates to room temperature overnight.

TAP (tris-acetate-phosphate) media

375 μM NH₄Cl, 17.5 μM CaCl₂·2H₂O, 20 μM MgSO₄·7H₂O, 6 μM Na₂HPO₄, 4 μM KH₂PO₄, 200 μM Trizma base, 170 μM glacial acetic acid, with Hutner’s trace elements solution at final concentration of 134 nM Na₂EDTA·2H₂O, 770 nM ZnSO₄·7H₂O, 184 nM H₃BO₃, 26 nM MnCl₂·4H₂O, 18 nM FeSO₄·7H₂O, 7 nM CoCl₂·6H₂O, 5 nM CuSO₄·5H₂O, and 0.8 nM (NH₄)₆Mo₇O₂₄·4H₂O). Suspended in ultrapure water. For TAP + 2.5% proteose peptone, we added 2.5% proteose peptone purchased from Millipore (Cat No 82450).

AF6 media

2 mM MES, 8.13 μM Fe-citrate, 10.4 μM citric acid, 1.65 mM NaNO₃, 275 μM NH₄NO₃, 122 μM MgSO₄·7H₂O, 73.5 μM KH₂PO₄, 28.7 μM K₂HPO₄, 68 μM CaCl₂·2H₂O, with trace elements (13.4 μM Na₂EDTA·2H₂O, 3.63 μM FeCl₃·6H₂O, 0.91 μM MnCl₂·4H₂O, 0.38 μM ZnSO₄·7H₂O, 84.1 nM CoCl₂·6H₂O, 51.7 nM Na₂MoO₄·2H₂O) and vitamins (29.6 nM thiamine-HCl, 8.2 nM biotin, 5.9 nM pyridoxine-HCl, 0.74 nM cyanocobalamin). pH adjusted to 6.6. Suspended in ultrapure water.

Bristol

2.94 mM NaNO₃, 0.17 mM CaCl₂·2H₂O, 0.3 mM MgSO₄·7H₂O, 0.43 mM K₂HPO₄, 1.29 mM KH₂PO₄, and 0.43 mM NaCl. Suspended in ultrapure fresh water.

Modified Bold’s 3N (MB3N)

8.82 mM NaNO₃, 0.17 mM CaCl₂·2H₂O, 0.3 mM MgSO₄·7H₂O, 0.43 mM K₂HPO₄, 1.29 mM KH₂PO₄, 0.43 mM NaCl, with P-IV trace metals (12 μM Na₂EDTA·2H₂O, 2.16 μM FeCl₃·6H₂O, 1.26 μM MnCl₂·4H₂O, 222 nM ZnCl₂, 50.4 nM CoCl₂·6H₂O, 102 nM Na₂MoO₄·2H₂O) and vitamins (100 nM vitamin B12 (cyanocobalamin), 100 nM biotin, 1 µM thiamine). The final media has 4% pasteurized soil water supernatant (0.05 mM CaCO₃, 2.5% greenhouse soil, suspended in ultrapure water and purchased from UTEX). pH adjusted to 6.2. Suspended in ultrapure water.

M-N

1.7 mM Na₃C₆H₅O₇·2H₂O, 37 μM FeCl₃·6H₂O, 360 μM CaCl₂·2H₂O, 1.22 mM MgSO₄·7H₂O, 1.32 mM K₂HPO₄·3H₂O, with trace elements (16.2 μM H₃BO₃, 3.5 μM ZnSO₄·7H₂O, 1.8 μM MnSO₄·H₂O, 0.84 μM CoCl₂·6H₂O, 0.83 μM Na₂MoO₄·2H₂O, 0.28 μM CuSO₄·5H₂O). pH adjusted to 6.8. Suspended in ultrapure water.

Nutrient broth

3.0 g/L beef extract and 5.0 g/L peptone. Purchased from Millipore (Cat No N7519). Suspended in ultrapure water.

Soil extract

96% Bristol media and 4% pasteurized soil water supernatant (0.05 mM CaCO₃, 2.5% greenhouse soil, suspended in ultrapure water and purchased from UTEX).

Erdschreiber’s

2.3 mM NaNO₃, 67 μM Na₂HPO₄·7H₂O, 23.7 μM Na₂EDTA·2H₂O, 4.3 μM FeCl₃·6H₂O, 2.5 μM MnCl₂·4H₂O, 400 nM ZnCl₂, 100 nM CoCl₂·6H₂O, 200 nM Na₂MoO₄·2H₂O, 100 nM cyanocobalamin, and 50 μM HEPES. Suspended in synthetic seawater (409 mM NaCl, 53 mM MgCl₂·6H₂O, 28 mM Na₂SO₄, 10 mM CaCl₂·2H₂O, 1 mM KCl, 1 mM NaHCO₃, 1 mM KBr, 0.4 mM SrCl₂·6H₂O, 1 mM H₃BO₃, 3 mM NaOH, and 2 mM NaF [RICCA Chemical Company: R8363000]) and 4% pasteurized soil water supernatant (0.05 mM CaCO₃, 2.5% greenhouse soil, suspended in ultrapure water and purchased from UTEX).

Growth conditions and documentation

After spotting, we left the plates upright to dry overnight in a tent equipped with grow lights under 24 hours of illumination at ambient temperature. The next day, we inverted the plates and placed them in clear, plastic shoeboxes to maintain humidity and prevent desiccation. We kept the plates under constant illumination at ambient temperature throughout the experiment.

We recorded initial observations 12 days post-inoculation, with particular attention to the enhanced growth of CC-3707 on proteose peptone-supplemented medium. We captured images using an Azure gel documentation system with Cy5 settings to visualize chlorophyll fluorescence as a proxy for colony growth and viability.

We performed comprehensive plate documentation at 20 days and 47 days post-inoculation using a standardized imaging setup consisting of a mini-light box on its side with a cardboard aperture to maintain consistent lighting across all plates. We acquired images with a Google Pixel 6 smartphone camera.

Pub preparation

We assembled ​​the figures in Adobe Illustrator. We used Claude to help write code, reorganize text using a template, suggest wording ideas, and then chose which small phrases or sentence structure ideas to use, help copy-edit draft text to match Arcadia's style, help clarify and streamline text that we wrote, and write text that we edited. We generated the Figure 1 heatmaps by converting concentrations to micromolar values using Python (v3.12.5) with NumPy (v2.1.2) and Pandas (v2.2.3) and plotted with Matplotlib (v3.9.2), Seaborn (v0.13.2), and arcadia-pycolor (v0.5.1) [13].

The observations

Our comparative growth analysis revealed distinctive phenotypic differences among the tested strains that appear unrelated to their characterized flagellar defects.

Enhanced growth on proteose peptone-supplemented medium

When we grew strains on standard TAP medium, we observed that the cpc1-1 and ida4 mutants exhibited a reduced colony density compared to wild-type CC-124 based on the chlorophyll intensity in the colonies (Figure 2, left). However, we observed a striking pattern reversal for cpc1-1 cells when we supplemented the medium with 2.5% proteose peptone (TAP+PP). On TAP+PP medium, cpc1-1 colonies displayed substantially enhanced growth compared to wild-type CC-124 cells, achieving greater colony diameter and density than wild-type strains under the same conditions.

This differential response became evident within 12 days of inoculation and was pronounced by day 20 (Figure 3), as documented in our systematic imaging. Of note, the peptone in the media gave the agar a dark yellow hue. The cpc1-1 mutant showed growth even at the 1:10 and 1:100 dilutions on TAP+PP, whereas wild-type strains and the ida4 mutant showed little to no growth. The enhanced growth was consistent across all three replicates.

Retention of chlorophyll production on alternative nitrogen sources

We identified a second distinctive phenotype on media containing nitrate as the primary nitrogen source (AF6, Bristol, modified Bold's 3N, and soil extract media), in contrast to TAP medium, which uses ammonium as its nitrogen source. Wild-type C. reinhardtii CC-124, CC-5415, and the ida4 mutant exhibited pronounced chlorosis on the nitrate-containing media, developing yellow colonies characteristic of strains with the nit2 mutation in the 137c lineage. This mutation impairs nitrate utilization, leading to nitrogen starvation and chlorophyll degradation (Figure 3) [12].

In contrast, cpc1-1 colonies maintained a dark-green coloration on these nitrate-containing media, similar to wild-type C. smithii (CC-1373), which has functional nitrate assimilation pathways. This unexpected chlorophyll retention suggests that cpc1-1 may have kept functional NIT2 alleles from its CC-2677 ancestry, enabling more efficient nitrogen utilization from nitrate sources.

Halotolerance of cpc1-1 on marine media

We discovered a third unexpected phenotype after extended growth periods. At 47 days post-inoculation, we observed that cpc1-1 was the only strain capable of growing on Erdschreiber's marine medium with elevated salt content (Figure 4) (Figure 1). While all other strains showed no detectable growth on this medium, cpc1-1 established a visible colony on two of the three plates, indicating a previously uncharacterized halotolerance phenotype. Though we've previously reported C. smithii growth on Erdschreiber's medium [10], we didn’t observe that here after 47 days. So it’s worth considering that colonies from other strains might appear on these plates over a more extended period, or if we'd used a denser starting culture, since we’d set these experiments up with dilute starter cultures.

This finding further supports that genetic elements in the cpc1-1 strain differ from the standard 137c background and confer adaptive advantages under specific environmental conditions.

Thoughts and questions

Our comparative growth analysis reveals that the cpc1-1 mutant (CC-3707) has three distinct phenotypes that differentiate it from the wild-type strains CC-124 and CC-5415 and the ida4 mutant: enhanced growth on protein-rich media, maintained chlorophyll production when using nitrate as a nitrogen source, and unique halotolerance on marine medium. These characteristics could stem from genetic elements inherited from the original CC-2677 (nit1-305) background that persisted through backcrossing into the 137c lineage. However, none of these phenotypes were present in CC-5415, the product of a cross between nit1-305 and a 137c background. Since CPC1 is known to interact with enolase and components of the metabolic machinery [5], it remains possible that the cpc1-1 allele causes these phenotypes; however, our current evidence isn’t sufficient to draw conclusions.

To address this, we’ll be taking a two-pronged approach. We’ve acquired strain CC-5148, which is derived from 137c strain CC-125 that spontaneously acquired a missense mutation preventing CPC1 protein from localizing to the flagella [14]. This mutant allele was named cpc1-2. We'll perform comparative studies on the growth of CC-5148 to determine if the cpc1-2 mutation is sufficient to induce these growth phenotypes in a controlled background. However, since the mutation appears only to impact CPC1 localization, the protein may still be able to function in the cell body in this mutant, so CPC1-dependent metabolic phenotypes might not be apparent. As a second approach, we’ll overexpress wild-type full-length CPC1 in both mutants to recover wild-type behavior independent of strain background.

• Acknowledgements
  • Thank you to Hayley Greenough for providing the agar media plates used in this study.