An Improved Natural Transformation Protocol for the Cyanobacterium Synechocystis sp. PCC 6803

Abstract

The naturally transformable cyanobacterium Synechocystis sp. PCC 6803 is a widely used chassis strain for the photosynthetic production of chemicals. However, Synechocystis possesses multiple genome copies per cell which means that segregating mutations across all genome copies can be time-consuming. Here we use flow cytometry in combination with DNA staining to investigate the effect of phosphate deprivation on the genome copy number of the glucose-tolerant GT-P sub-strain of Synechocystis 6803. Like the PCC 6803 wild type strain, the ploidy of GT-P cells grown in BG-11 medium is growth phase dependent with an average genome copy number of 6.05 ± 0.27 in early growth (OD 740 = 0.1) decreasing to 2.49 ± 0.11 in late stationary phase (OD 740 = 7). We show that a 10-fold reduction in the initial phosphate concentration of the BG-11 growth medium reduces the average genome copy number of GT-P cells from 4.51 ± 0.20 to 2.94 ± 0.13 and increases the proportion of monoploid cells from 0 to 6% after 7 days of growth. In addition, we also show that the DnaA protein, which unusually for bacteria is not required for DNA replication in Synechocystis, plays a role in restoring polyploidy upon subsequent phosphate supplementation. Based on these observations, we have developed an alternative natural transformation protocol involving phosphate depletion that decreases the time required to obtain fully segregated mutants.

The Genome Copy Number of the Thermophilic Cyanobacterium Thermosynechococcus elongatus E542 Is Controlled by Growth Phase and Nutrient Availability

“…Flow cytometry was performed using Attune NxT Flow Cytometer as described previously (12). Briefly, ethanol fixed cells were washed twice with PBS and incubated with RNAse A (1µg/ml) at 37 °C for hour.…”

“…Others, like Caulobacter crescentus are monoploid irrespective to the growth rate (9). Alternatively, some bacteria maintain multiple chromosome copies per cell (polyploidy) irrespective of growth rate as observed in many cyanobacteria (10)(11)(12)(13). Ploidy of cyanobacteria is highly variable, it is influenced by growth phase, chemical and physical parameters and stress conditions (10,12,14,15).…”

“…Alternatively, some bacteria maintain multiple chromosome copies per cell (polyploidy) irrespective of growth rate as observed in many cyanobacteria (10)(11)(12)(13). Ploidy of cyanobacteria is highly variable, it is influenced by growth phase, chemical and physical parameters and stress conditions (10,12,14,15). Previous studies have discussed the various benefits of polyploidy in prokaryotes (16) including low mutation rate, resistance against double-strand breaks, gene redundancy, global regulation of gene dosage, large cell size and storage of phosphate.…”

“…Previous studies have discussed the various benefits of polyploidy in prokaryotes (16) including low mutation rate, resistance against double-strand breaks, gene redundancy, global regulation of gene dosage, large cell size and storage of phosphate. However, polyploid cells impede the on May 10, 2021 by guest http://aem.asm.org/ Downloaded from construction of mutants, and segregating desired mutations across all genome copies are timeconsuming, often taking more than a month (12). Thermophilic cyanobacteria of the family Thermosynechococacceae, including recently isolated Thermosynechococcus elongatus E542 (FACHB-2455) are underexplored for their genetics compared to cyanobacterial model strains.…”

“…Previous studies have discussed the various benefits of polyploidy in prokaryotes ( 16 ), including the low mutation rate, resistance against double-strand breaks, gene redundancy, global regulation of gene dosage, large cell size, and storage of phosphate. However, polyploid cells impede the construction of mutants, and segregating desired mutations across all genome copies is time-consuming, often taking more than a month ( 12 ).…”