Chitin degradation by Synechococcus WH7803

Abstract

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.

Figure 1

Mutants lacking chiA or 2345 obtained with a CRISPR-Cpf1 approach. (a) Cartoon representation of the WH7803 genome and the relative positions of the genes of interest. (b) Schematic representation of the edited cell lines 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 Table S2, which are designed outside the homologous template regions.

Figure 2

Rate of chitin degradation activity in mutants lacking ChiA or 2345 in comparison to wild-type WH7803. (a, b) Expression (measured by qPCR) of 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. (c, d) 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 < 0.0, ns not significant using Welch’s t test). Activity is lost after boiling and shown as a negative control for each sample.