Figure 3. Expression of 20 CCM genes permits growth of CCMB1 in ambient air.

Time course data give representative growth curves from a bioreactor bubbling ambient air. CCMB1:pCB’ + pCCM’ grows well (purple, ‘full CCM’), while rubisco and prk alone are insufficient for growth in ambient air (grey, CCMB1:p1A+vec). Inset: a plate reader experiment in biological triplicate (different shades) gave the same result. Expressing the full complement of CCM genes led to an increase in culture density (optical density at 600 nm) of ≈0.6 units after 80 hr of cultivation. Bootstrapping was used to calculate a 99.9% confidence interval of 0.56–0.64 OD units for the effect of expressing the full CCM during growth in ambient air. Figure 3—figure supplement 1 and Appendix 2 describe the selection procedures in detail while Figure 3—figure supplement 2 shows triplicate growth curves and evaluates statistical significance.

Figure 3—figure supplement 1. A series of selection experiments produced mutant plasmids that permit rubisco-dependent growth in ambient air.

(A) pCB and pCCM plasmids together encode 20 H. neapolitanus genes including 12 confirmed CCM components. pCB carries kanamycin resistance and has two transcriptional units both expressed under an aTc-inducible P_LtetO-1 promoter (Lutz and Bujard, 1997). The first derives from pHnCB10 (Bonacci et al., 2012) and expresses 10 carboxysome proteins. The second expresses phosphoribulokinase (prk). pCCM carries chloramphenicol resistance and expresses an 11 gene operon from H. neapolitanus that contains both putative and confirmed CCM genes (Desmarais et al., 2019). Although pCB expresses both rubisco and prk, CCMB1:pCB did not initially grow in M9 media under 10% CO₂ (not shown) and so we undertook a series of selections, described in panels (B–D) that ultimately led to isolation of pCB’ and pCCM’ plasmids that together enable CCMB1 to grow in ambient air. (B) We first selected CCMB1:pCB for growth on minimal media by screening for mutants able to grow on M9 glycerol and then M9 gluconate media. Gluconate growing mutant #9 (gg.9) was used for subsequent experiments as this mutant was found to grow best on gluconate (as shown in E). (C) Plasmid extracted from gg.9 was deep sequenced and electroporated into naive CCMB1 to test for plasmid linkage of growth on minimal. (D) Selection for rubisco-dependent growth in ambient air. A turbid pre-culture of the CCMB1:pCB gg.9+pCCM double transformant was washed and plated on M9 glycerol media under ambient air. Colonies formed after ≈20 days (as shown in F). Forty colonies (s.1–40) were picked into rich media, grown to saturation, washed and plated on M9 glycerol media to verify growth under ambient air. Roughly ¼ of chosen colonies regrew under ambient air to varying degrees (s.1–6 are shown in G). Plasmid extracted from several strains was deep-sequenced and electroporated into naive CCMB1 to test plasmid-linkage of growth on glycerol minimal media in ambient air. Pooled plasmid extracted from s.4 was found to confer replicable growth in ambient air (as shown in H). PCR and Gibson cloning were used to reconstruct the individual pCB and pCCM plasmids from this pool. We termed these reconstructed plasmids pCB’ and pCCM’. (E) Restreaking of gluconate-growing mutants gg.8–12 described in panel B shows that gg.9 grew best on gluconate. (F) CCMB1:pCB gg.9+pCCM double transformants were plated for mutants on M9 glycerol media under ambient air. A negative control lacking carboxysome genes (CCMB1:p1A+pCCM) was plated at the same time. Colonies formed after 20 days (bottom right) only on induced plates (100 nM) and only when all CCM genes were provided (i.e. pCB gg.9 and pCCM). (G) Several of the chosen colonies regrew in ambient air. Growth characteristics varied from colony to colony, suggesting genetic variation. (H) Pooled plasmid extracted from s.4 was found to permit naive CCMB1 to grow in ambient air. For comparison, plasmid from s.6 produced less reproducible growth in ambient air.

Figure 3—figure supplement 2. pCB’ and pCCM’ permit CCMB1 to grow in ambient air.

(A) Biological triplicate growth curves from a bioreactor bubbling ambient air. CCMB1 co-transformed with post-selection plasmids pCB’ and pCCM’ (CCMB1:pCB’ + pCCM’) grows well (purple, ‘full CCM’), while rubisco and prk alone are insufficient for growth in air (grey, ‘rubisco+prk’). Maximal growth rates for the 'full CCM' cultures ranged from 0.03 to 0.06 hr⁻¹, corresponding to doubling times of 12–25 hr. As these are biological replicate cultures, heterogeneity in growth kinetics could be due to genetic effects (e.g. point mutations in founding colonies) or non-genetic differences (e.g. varying degree of carboxysome production during pre-culturing). (B) Data for the same strains grown in a 96-well plate in ambient air in a shaking plate reader. Different shades mark biological replicates (pre-cultures deriving from three distinct colonies). Additionally, each preculture was used to inoculate at least 12 technical replicates. (C) Quantification of the experiment in panel (B) using endpoint data at 80 hr for biological and technical replicates. Panel (C) uses the same colors as (A) and (B) with the addition of a rubisco active site mutant as a negative control (grey, CCMB1:p1A^- + vec). ‘****’ indicates p<10⁻¹⁰. p-Values were calculated with a Bonferroni-corrected two-sided Mann-Whitney-Wilcoxon test. 10⁴-fold bootstrapping was used to compare ‘full CCM’ data to ‘rubisco + prk’ and estimate a confidence interval for the effect of expressing a full CCM on growth in ambient air, which gave a 99.9% confidence interval of 0.56–0.64 OD units.