Cyanobacteria, also known as blue-green algae, are photosynthetic prokaryotes. They played a key role in the evolution of life on Earth, converting the early reducing atmosphere into an oxidizing one as they performed oxygenic photosynthesis . Cyanobacteria are thought to be progenitors of chloroplasts via endosymbiosis . Approximately, 20–30% of Earth's photosynthetic activity is due to cyanobacteria. The proteomic composition and dynamics of plasma membranes of cyanobacteria have been extensively characterized [2, 3]. However, the influence of the structure and composition of cyanobacterial membranes on cellular uptake remains largely unknown. Delivery of exogenous DNA into cyanobacteria was first reported in 1970 , although the internalization mechanisms are still unknown . Since cyanobacteria play key roles in supporting life on Earth and have potential in biofuel production and other industrial applications [5–7], understanding how they interact with the environment by processes such as internalization of exogenous materials, is becoming increasingly important.
The plasma membrane provides a barrier that hinders the cellular entry of macromolecules, including DNAs, RNAs, and proteins. In 1988, two groups simultaneously identified a protein called transactivator of transcription (Tat) from the human immunodeficiency virus type 1 (HIV-1) that possesses the ability to traverse cellular membranes [8, 9]. The penetrating functional domain of the Tat protein is comprised of 11 amino acids (YGRKKRRQRRR) . Subsequently, many peptide analogues of the basic amino acid-rich domain of the Tat protein were synthesized and evaluated for membrane transduction potential [11, 12]. These positively charged, amphipathic peptides were termed cell-penetrating peptides (CPPs) or protein transduction domains (PTDs) [11–13].
Among synthetic peptides, the cellular uptake of polyarginine was found to be much more efficient than that of polylysine, polyhistidine, or polyornithine [13, 14]. We found that a nona-arginine (R9) CPP peptide can enter cells by itself or in conjunction with an associated cargo [15–21]. Cargoes that R9 can carry include proteins, DNAs, RNAs, and inorganic nanoparticles (notably, quantum dots; QDs). R9 can form complexes with cargoes in covalent, noncovalent, or mixed covalent and noncovalent manners [22–24]. CPPs can deliver cargoes up to 200 nm in diameter [11, 25], and R9 can internalize into cells of various species, including mammalian cells/tissues, plant cells, bacteria, protozoa, and arthropod cells [16, 17, 26, 27].
Despite many studies using various biological and biophysical techniques, our understanding of the mechanism of CPP entry remains incomplete and somewhat controversial. Studies have indicated that CPPs enter cells by energy-independent and energy-dependent pathways . The concentration of CPPs appears to influence the mechanism of cellular uptake . Our previous studies indicated that macropinocytosis is the major route for the entry of R9 carrying protein or DNA cargoes associated in a noncovalent fashion [15, 29, 30]. However, we found that CPP/QD complexes enter cells by multiple pathways [31, 32]. Multiple pathways of cellular uptake were also demonstrated with CPP-fusion protein/cargo complexes associated in a mixed covalent and noncovalent manner [22, 24]. In contrast, our study of the R9 modified with polyhistidine (HR9) indicated direct membrane translocation .
The cellular entry mechanisms of CPPs in cyanobacteria have not been studied. In the present study, we determined CPP-mediated transduction efficiency and internalization mechanisms in cyanobacteria using a combination of biological and biophysical methods.