E. coli DH5α was used for all plasmid manipulation using standard procedures. S. elongatus PCC 7942 was cultured in BG-11 medium [31] at 30°C and illuminated by strong light. CHO and J774 cells were maintained using standard procedure in F-12 medium (Invitrogen) for CHO cells and RPMI 1640 medium (Invitrogen) for J774 cells. All media contained L-glutamine and were supplemented with 10% FBS (HyClone) and 1% Penicillin/Streptomycin Mix (Invitrogen). For culturing cells during infections outside of the controlled 5% CO2 atmosphere, Leibovitz's L-15 medium without phenol red (Invitrogen) was used for all cell lines, supplemented with 10% FBS for all cell types and 0.069 mg/ml proline for CHO cells.

The invasin gene from Yersinia pestis (inv) was subcloned from the pAC-TetInv plasmid [16] provided by Chris Voigt (University of California, San Franscisco) and listeriolysin O (llo) was amplified from Listeria monocytogenes genomic DNA provided by Heather Kamp (Harvard Medical School, Boston MA). Invasin DNA was amplified with primers adding a SpeI site upstream (5′-CGCAACTAGTATGGTTTTCCAGCCAATCAG-3′) and NotI and XbaI sites downstream (5′-CTGCAGCGGCCGCTAGCTCTAGATTATATTGACAGCGCACAGA-3′) Listeriolysin was amplified with primers adding a SpeI site, a ribosome binding site, and a short spacer for cloning downstream of invasin (5′-CGCAACTAGTAGGAGGAAAAACATATGAAAAAAATAATGCTAGTTTT-3′) and NotI and XbaI site downstream (5′-CTGCAGCGGCCGCTTCTAGATTATTCGATTGGATTATCTA-3′). Invasin and listeriolysin were then sequentially subcloned into the pNS3 vector for homologous recombination into Synechococcus neutral site 3 [30].

The pNS3-invllo vector was incubated overnight in the dark with a culture of S. elongatus PCC 7942 cells washed in 10 mM NaCl, and integration into the neutral site was selected using BG11 plates containing 1.5% Noble Agar and 12.5 µg/ml chloramphenicol. Expression of invasin and listeriolysin was induced with 100 µM IPTG for 24 hours.

Zebrafish embryos in the one-cell stage were injected with a solution containing mRNA for expression of membrane GFP (mGFP) and bacteria. Needles were pulled on a Sutter P2000 laser needle puller from Drummond glass capillary tubes. Eggs were injected with 2.3 nl of injection solution using a Nanoject. The injection solution consisted of injection buffer (50 mM NaCl, 1 mM Tris pH 8, 0.1 mM EDTA and 0.1% Phenol Red) and contained 40 ng of mGFP mRNA and 1 µl of a saturated bacterial suspension per 10 µl of injection solution. The bacterial suspension (S. elongatus or E. coli) was prepared by spinning down 1 ml of an overnight E. coli culture or a dense 24–48 h old, exponentially growing S. elongatus culture. The cells were resuspended in 1 ml of injection buffer without Phenol Red and again pelleted. The supernatant was removed; the cells were mixed and always used fresh for the injections.

Embryos were raised in egg water (0.3 g/L Instant Ocean, 75 mg/L CaSO₄) slightly shaded from the light in the cyanobacterial incubator at 30°C. Egg water was changed as needed. To follow individual embryos over time, the embryos were separated from each other in 12-well plates.

Development of the embryos injected with bacteria was monitored with a fluorescence dissecting microscope. For confocal imaging the embryos were dechorionated and placed in imaging molds made from 1% (w/v) agarose in egg water. Mounted embryos were imaged in an upright Zeiss LSM 710 confocal microscope. The embryo was submerged in eggwater containing 1× Tricaine solution (10× Tricaine solution: 0.1% (w/v) Tricaine and 10 mM Tris in egg water adjusted to pH 7 with NaOH) for anesthetization.

All of our zebrafish protocols were approved by the Harvard Medical School (HMS) Office for Research Subject Protection and the HMS Standing Committee on Animals (IACUC Approval Number 04487).

For infections of mammalian cells with bacteria, induced bacteria were washed and transferred from their culture medium into PBS (137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 2 mM KH2PO4). Bacterial suspensions in PBS were set to the same OD and 100 µl of this suspension were added per 2 ml of cell culture medium per well of 12-well tissue culture dishes containing the mammalian cells. L-15 medium during the infection did not contain antibiotics. After the treatment of the cells with S. elongatus for 3 hours to overnight, the cells were washed with PBS three times and the medium was replaced by L-15 containing 100 µg/ml gentamicin, an antibiotic that does not cross the mammalian cell membrane. During and after infections of the mammalian cells with bacteria, the cultures were kept at 30°C in atmospheric CO₂ levels. For S. elongatus, cells were illuminated with fluorescent lamps from both sides of the tissue culture plate.

For time-course of S. elongatus infection in macrophages, 10,000 J774 cells were plated per well of a 96-well plate in L-15 media and were allowed to attach to the bottom overnight at 37°C in the dark. Following attachment, 10 microliters of wild type or inv/llo engineered S. elongatus diluted to OD₇₅₀ of 0.025–0.4 in PBS (corresponding to approximately <1 bacteria per macrophage to >4 bacteria per macrophage) were added to each well and incubated at 30°C in the light for six hours. Each well was then washed in PBS and the media was replaced with L-15 containing 100 µg/ml gentamicin and plates were incubated at 30°C in the light. One plate was removed every 24 hours and cells were fixed in 3% paraformaldehyde, cells were permeablized in 0.01% Triton-X in PBS and stained with DAPI. Plates were stored at 4°C in the dark and imaged at the same time using fluorescence microscopy.

After 24 hour infection, CHO cells were washed in PBS, trypsinized, and resuspended in FACS buffer (PBS supplemented with 1%FBS). Cells were sorted with a BD FACSAria cell sorter based on red channel fluorescence. Cells positive for red fluorescence were gently reattached to glass-bottomed tissue culture dishes with concanavalin A and imaged with confocal microscopy.

Photosynthetic symbiosis exists in several underwater species, such as the sea slug Elysia chlorotica, which incorporates the chloroplasts from algae that it feeds on into the cells of its intricately branched digestive system, allowing it to survive for months photoautotrophically [7]. While such a complex symbiotic relationship likely evolved over much longer time scales, we were interested in replicating the first step of an underwater photosynthetic symbiosis and exploring the in vivo dynamics of photosynthetic bacteria in a developing animal. We chose zebrafish embryos as they are easy to microinject, well studied, and are clear, allowing light to penetrate.

Up to ten million bacteria were injected into zebrafish embryos at the single cell stage to track the relationship between the vertebrate and bacterial cells through development. Red autofluorescent bacteria were found intracellularly throughout the embryo during development, including in the brain and even the lens of the eye (figure 2A–D) with no discernible morphological effects. Synechococcus survived inside the embryo's cells for up to twelve days based on continued red autofluorescence (figure 2F), at which time the experiment was terminated as the fish began to develop pigment that would block light to the intracellular bacteria.

In stark contrast, injecting E. coli cells killed the embryo within two hours (figure 3B). This rapid death occurred even when the E. coli were UV killed prior to injection (figure 3C) and when the Lipid A production was attenuated by deletion of msbB (figure 3D), a modification shown to decrease incidence of septic shock by tumor-targeting Salmonella [32]. These data point to other surface markers that can cause the death seen in the E. coli injected embryos and a benign role for the non-pathogenic S. elongatus in vivo.

We also sought to explore a more physiological model of intracellular invasion than direct microinjection. The bacterial virulence factors encoding mammalian cell invasion—invasin from Yersinia pestis [33]—and escape from the lysosomal compartment—listeriolysin O from Listeria monocytogenes [34] have been identified, cloned, and shown to confer invasive behavior to non-pathogenic bacterial species. We inserted invasin and listeriolysin as a tandem operon in S. elongatus neutral site 3 [30] and incubated induced S. elongatus cells with CHO cells at 50%–80% confluence overnight at 30°C in bright light.

While expression of invasin alone is sufficient for high efficiency invasion by E. coli for multiple mammalian cell types in culture (HeLa, U2OS, HepG2, CHO [16]), expression of both invasin and listeriolysin was required for invasion of CHO cells by S. elongatus, a result previously reported for engineered E. coli cells invading colon cancer cells [23]. The invasion efficiency was such that 4.8% of mammalian cells were positive for the red channel autofluorescence from intracellular photosynthetic bacteria as measured by fluorescence-activated cell sorting (FACS) analysis (figure 4A). Sorted cells were imaged with confocal microscopy (figure 4B), confirming intracellular localization with approximately one bacterial cell per CHO cell analyzed.

Bacteria can also enter cells through phagocytosis, and escaping digestion by the lysosome is a prerequisite for pathogenic and symbiotic growth. Macrophages are a crucial part of the mammalian immune system, seeking out, engulfing and digesting foreign bodies and bacteria. The immortal mouse macrophage cell line J774 will quickly engulf large numbers of bacterial cells in culture. We therefore incubated plates of 50% confluent macrophages with varying concentrations of bacterial cells for one hour at 37°C for E. coli and six hours at 30°C for S. elongatus. As with zebrafish embryos, engulfed E. coli cells will quickly kill their host macrophages (figure 5A), while even high numbers of S. elongatus cells will remain inside J774 for several days with relatively little effect.

E. coli highly expressing listeriolysin were observed to kill macrophages faster than wild type E. coli (figure 5B). However, after two days of incubation with Synechococcus, similar levels of cell death were observed in macrophages with Synechococcus with only the empty vector integrated (figure 5C), those expressing invasin and listeriolysin (figure 5D), and with macrophages without any bacteria (figure 5E).

Non-pathogenic bacteria that have been engineered with listeriolysin O to escape the macrophage endosome have been shown to replicate in the cytoplasm [25]. However, there are many factors in the mammalian cytoplasm speculated to be involved in preventing bacterial growth, a fact suggested by the extremely small number of intracellular pathogens able to divide in the presumably nutrient-rich cytoplasm [24]. S. elongatus requires little external metabolic input and grows at a relatively fast rate at intracellular carbon dioxide concentrations (one division every 8–12 hours). In addition, as we have shown (above) S. elongatus has a special relationship to eukaryotic antimicrobial systems as it is able to peacefully coexist with animal cells. As such, it is expected that S. elongatus engineered with listeriolysin O will be able to divide in the mammalian cytoplasm.

In the dark, S. elongatus phogocytosed by macrophages will rapidly lose red channel autofluorescence over the course of 12 hours, indicating death (figure 6A). In the light, wild type S. elongatus autofluorescence will more slowly decrease in intensity over several days (figure 6B, top row). S. elongatus engineered with invasin and listeriolysin, able to escape lysosome digestion, showed marked increase in autofluorescence in the first two days post-infection, with the number of autofluorescent bacteria decreasing only after 3 days (figure 6B, bottom row).

The rate of division in the macrophage cytoplasm was quantified for varying densities of S. elongatus in 96 well plates. Mean, background subtracted fluorescence was averaged for triplicate infections. At similar starting density of approximately 2 bacteria per macrophage, there is marked contrast between empty vector (blue) and +inv+llo (red) S. elongatus (figure 6C). Rates of division in the engineered strain were correlated to S. elongatus infection densities. At the lowest concentrations, with fewer than one bacterial cell per macrophage, the engineered strain is digested more slowly than wild type S. elongatus, but does not show large-scale evidence of division, but as infection density is doubled, the rate of growth increases and begins to level off at the highest density (1–4 bacteria per macrophage, figure 6D). Differences in S. elongatus growth did not correlate with decrease in macrophage cell counts over time, which remained variable but consistent between wells at different infection densities over time (figure 6E). Even at the lowest infection density, +inv+llo S. elongatus division was observed in approximately 1% of cells tracked with time-lapse microscopy (figure 6F).