Chitin degradation by Synechococcus WH7803

Chitin is an abundant, carbon-rich polymer in the marine environment. Chitinase activity has been detected in spent media of Synechococcus WH7803 cultures—yet it was unclear which specific enzymes were involved. Here we delivered a CRISPR tool into the cells via electroporation to generate loss-of-function mutants of putative candidates and identified ChiA as the enzyme required for the activity detected in the wild type.

Because of the difference in size, the wild-type band in ∆2069 is non-detectable with this set of primers.c, Expression (measured by qPCR) of 2069, chiA, or 2345 in wild-type and mutant lines in mid-exponential growth in relation to the housekeeping gene, rnpB, in natural seawater-based Pro99 medium in presence and absence of colloidal chitin.Expression of 2069 in ∆2069 was significantly lower than in the WT (*P < 0.05, **P < 0.01, ns= not significant using Welch's t-test).
Fig. S3: Growth rates in Synechococcus WT and mutant lines.Growth of Synecococcus WH7803 WT and recovered mutant lines in continuous light (at 12 μmol photons m −2 s −1 ) monitored by relative bulk culture chlorophyll fluorescence.Different colors show the average growth for each line in Pro99 media or Pro99 media amended with colloidal chitin.Growth rates and associated standard deviation (μ, in units day -1 ) was calculated in exponential phase (marked with a black line) and is shown for each curve.

Fig.S4: Chitin degradation activity
Endochitinase and Exochitinase (chitobiosidase) activities measured in wild-type and mutant lines spent media amended with either colloidal chitin or chitosan to a final concentration of 56 μg/ml.Average and statistical significance of activities obtained from chitosan or colloidal chitin addition are shown in red and maroon, respectively (*P < 0.05, **P < 0.01, ns= not significant using Welch's ttest).Activity is lost after boiling and shown as a negative control for each sample.Data relative to WT, ∆chiA, and ∆2345 is also reported in Figure 2a-b.

Fig.S5: Adhesion of cells to colloidal chitin particles
Cultures were grown in Pro99 media with (solid line) and without (dashed line) added colloidal chitin.Growth was monitored by bulk chlorophyll fluorescence a-d, and cells in suspension were measured using flow cytometry e-h.Error bars show standard deviation between three biological replicates.The average growth rate and associated standard deviation (μ, in units day -1 ) was calculated in exponential phase (marked with a black line) and is shown for each curve.While growth rates between treatments did not differ, Synechococcus cells in suspension were less abundant in presence of chitin, as cells attaching to the polymer avoid detection via flow-cytometer.MED4, a high-light adapted Prochlorococcus ecotype, does not stick to chitin 2 , and consistently no difference was found in cells in suspension between the two treatments.(*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 using Welch's t-test)

Fig. S2 :
Fig.S2: Mutant lacking 2069 a, Cartoon representation of the WH7803 genome and the relative positions of all the three genes of interest.b, Schematic representation of the edited cell line obtained with the CRISPR-Cpf1 tool.Sanger sequences show the details of each deletion.Orange circles show the location of the PAM sites.PCR products indicate the length of each amplification using primers listed in TableS2.Because of the difference in size, the wild-type band in ∆2069 is non-detectable with this set of primers.c, Expression (measured by qPCR) of 2069, chiA, or 2345 in wild-type and mutant lines in mid-exponential growth in relation to the housekeeping gene, rnpB, in natural seawater-based Pro99 medium in presence and absence of colloidal chitin.Expression of 2069 in ∆2069 was significantly lower than in the WT (*P < 0.05, **P < 0.01, ns= not significant using Welch's t-test).