An easy and efficient permeabilization protocol for in vivo enzyme activity assays in cyanobacteria.
BACKGROUND
Cyanobacteria are photosynthetic bacteria that thrive in diverse ecosystems and play major roles in the global carbon cycle. The abilities of cyanobacteria to fix atmospheric CO2and to allocate the fixed carbons to chemicals and biofuels have attracted growing attentions as sustainable microbial cell factories. Better understanding of the activities of enzymes involved in the central carbon metabolism would lead to increasing product yields. Currently cell-free lysates are the most widely used method for determination of intracellular enzyme activities. However, due to thick cell walls, lysis of cyanobacterial cells is inefficient and often laborious. In some cases radioisotope-labeled substrates can be fed directly to intact cells; however, label-free assays are often favored due to safety and practical reasons.


RESULTS
Here we show an easy and highly efficient method for permeabilization of the cyanobacteria Synechococcus sp. PCC 7002 and Synechocystis sp. PCC 6803, and determination of two intracellular enzymes, ribulose-1,5-bisphosphate carboxylase/decarboxylase (Rubisco) and glucose-6-phosphate dehydrogenase (G6PDH), that play pivotal roles in the central carbon metabolism in cyanobacteria. Incubation of the cyanobacterial cells in the commercially available B-PER reagent for 10 min permeabilized the cells, as confirmed by the SYTOX Green staining. There was no significant change in the cell shape and no major loss of intracellular proteins was observed during the treatment. When used directly in the assays, the permeabilized cells exhibited the enzyme activities that are comparable or even higher than those detected for cell-free lysates. Moreover, the permeabilized cells could be stored at -20 °C without losing the enzyme activities. The permeabilization process and subsequent activity assays were successfully adapted to the 96-well plate system.


CONCLUSIONS
An easy, efficient and scalable permeabilization protocol was established for cyanobacteria. The permeabilized cells can be directly applied for measurement of G6PDH and Rubisco activities without using radioisotopes and the protocol may be readily adapted to studies of other cyanobacterial species and other intracellular enzymes. The permeabilization and enzyme assays can be performed in 96-well plates in a high-throughput manner.
Department of Plant and Environmental Sciences, Copenhagen Plant Science Center, University of Copenhagen, Frederiksberg, Denmark.
Randi Engelberth
RE
Rasmussen
Department of Plant and Environmental Sciences, Copenhagen Plant Science Center, University of Copenhagen, Frederiksberg, Denmark.
Simon Matthé
SM
Erstad
Department of Plant and Environmental Sciences, Copenhagen Plant Science Center, University of Copenhagen, Frederiksberg, Denmark.
Erick Miguel
EM
Ramos-Martinez
Department of Plant and Environmental Sciences, Copenhagen Plant Science Center, University of Copenhagen, Frederiksberg, Denmark.
Lorenzo
L
Fimognari
Department of Plant and Environmental Sciences, Copenhagen Plant Science Center, University of Copenhagen, Frederiksberg, Denmark.
Alice Jara
AJ
De Porcellinis
Department of Plant and Environmental Sciences, Copenhagen Plant Science Center, University of Copenhagen, Frederiksberg, Denmark. ysa@plen.ku.dk.
Yumiko
http://orcid.org/0000-0002-9405-5197
Y
Sakuragi
D001426:Bacterial Proteins / Q000378:metabolism*; D002463:Cell Membrane Permeability; D005954:Glucosephosphate Dehydrogenase / Q000378:metabolism*; D010539:Permeability; D012273:Ribulose-Bisphosphate Carboxylase / Q000378:metabolism*; D046940:Synechococcus / Q000201:enzymology*; D046939:Synechocystis / Q000201:enzymology*
D001426:Bacterial Proteins; D005954:Glucosephosphate Dehydrogenase; D012273:Ribulose-Bisphosphate Carboxylase
B-PER; Cyanobacteria; Enzyme activities; Glucose-6-phosphate dehydrogenase; Permeabilization; Rubisco; SYTOX Green