While the evolution of cooperation and altruism are often seen as paradoxical events in the course of natural selection, endosymbiosis has been recognized as a driver of evolutionary change. Not only has gene exchange been observed between hosts and symbionts 
, but the development of communities suitable to new ecological niches 
and even the origin of the eukaryotic kingdom hinge on symbiotic collaborations 
. Modern endosymbiotic relationships between bacteria and eukaryotic organisms reflect a remarkable diversity in how widely disparate species can interact in positive ways, from nutritional symbiosis between Buchnera
and aphids 
, to nitrogen fixation by Rhizobia
in plant root nodules 
and photosynthetic symbiosis between algal chloroplasts and sea slugs 
Cooperative behavior and symbiotic relationships are widespread in nature and have recently begun to be exploited in synthetic biological networks of increasing complexity 
. Multi-component synthetic-ecological systems have been developed for hydrogen production through metabolic engineering 
and for the production of other useful metabolites 
. Communication between cells has also been engineered for multiple applications, including pattern formation 
and oscillators 
. Engineered communities have also been useful as a generalized model of cooperation and competition in microbial populations 
and two-species metabolic modeling has been used in the identification of cooperating variants of E. coli 
. While invasive bacteria have been explored as tools for synthetic biology and targeted tumor killing bacteria 
, neutral or beneficial endosymbiosis has not been pursued.
There is a fine line between the pathological and beneficial in natural endosymbiotic events. Many endosymbiotic relationships that exist in nature are hypothesized to have begun through the acquisition of resistance to predation— bacterial resistance to lysosomal digestion by amoeba after phagocytosis or eukaryotic resistance to bacterial infection after intracellular invasion 
. Replicating these events in the laboratory may lead to a partial endosymbiosis. Symbiosis is generally thought to refer to a mutualistic relationship where both partners benefit, but the term can be construed rather broadly; Lynn Margulis paraphrases de Bary's 1879 definition of symbiosis as simply the “protracted physical associations among organisms of different species, without respect to outcome.” 
We explored three paths for entry of photosynthetic bacteria into animal cells that would satisfy this broad definition of symbiosis ()—direct microinjection into zebrafish embryos to explore the in vivo
dynamics in a whole animal model, engineering with invasin from Y. pestis
(inv) and listeriolysin O from L. monocytogenes
(llo) to allow invasion of mammalian endothelial cells, and endocytosis of inv and llo engineered strains by macrophages. Invasin is a bacterial surface protein that interacts with mammalian β1-integrins and causes uptake of the bacterial cells, while listeriolysin O is a hemolysin that disrupts the endosomal membrane and allows bacteria to enter the mammalian cytoplasm post-uptake.
Three paths to endosymbiosis used in this study.
Invasive bacteria cause several deadly infectious diseases in humans, caused by intracellular pathogens such as Y. pestis
, L. monocytogenes
, and enteroinvasive E. coli 
. Recent work in biological engineering and synthetic biology has focused on the development of non-infectious but invasive and deadly bacteria that target and destroy only specific cell types for disease treatment, particularly cancer 
, or for delivery of peptide 
or nucleotide based vaccines 
and RNA interference gene therapy 
Macrophages can take up and phagocytose many different species of bacteria. However, most species of bacteria, including many pathogens, are unable to replicate in the cytoplasm of mammalian cells, and the precise mechanism of growth inhibition is unknown and a matter of controversy 
. In contrast, non-pathogenic Bacillus subtilis
expressing heterologous hemolysin has been shown to escape phagosome digestion by macrophages and divide in the mammalian cytoplasm 
. However, microinjection studies have found that only those species that naturally divide in the cytoplasm were able to replicate upon injection into mammalian cells, with even intravacuolar pathogens unable to divide in the cytoplasm 
. To our knowledge, such experiments have not been attempted with photosynthetic bacteria or other autotrophs.
Nearly eighty years ago, photosynthetic algae were explored as symbionts for cells grown in tissue culture, as a method for renewing and replenishing growth media with oxygen and nutrients while removing waste products and carbon dioxide 
. More recently, photosynthetic symbiosis in tissue culture was explored as a method for understanding the nutritional requirements of host and symbiont 
. We sought to explore the behavior of the photosynthetic bacteria Synechococcus elongatus
inside eukaryotic cells as a platform for engineered photosynthetic endosymbiosis and found that cyanobacteria have little apparent effect on their host cells and can divide in the macrophage cytoplasm. Further engineering of metabolite production and secretion 
in such endosymbiotic strains has the potential to lead to true mutualistic relationships between photosynthetic bacteria and mammalian cells, essentially creating artificial, engineerable, animal chloroplasts.