Sample collection was performed during the summer solstice of 2013 and 2014 in a set of solar panels situated in several locations in Valencia (Spain) by Dorado-Morales et al. (2016). Briefly, sterile PBS was poured on the panel and the liquid was harvested by scraping the surface with a sterile window cleaner. During the sampling process, the average temperature of the panels’ surfaces was 51°C and the average solar irradiance in Valencia was 461.3 W m⁻². Samples were stored at −80°C until their use. Further detailed information about field collection and samples is provided in Dorado-Morales et al. (2016).

Standard methods were used for the isolation and cultivation of cyanobacteria. Samples were spread on plates (1.5% wt/vol Purified Agar) with different cyanobacterial media: BG11 (Rippka et al., 1979), Spirulina medium (hereafter UTEX, Starr and Zeikus, 1987), Castenholz-D medium (Mühlenhoff and Chauvat, 1996), and MDM (Watanabe, 1960; Supplementary Table S1). BG11 and MDM were also used without nitrogen combined sources (BG11₀ and MDM₀, respectively). As cyanobacteria from environmental samples often grow more slowly than other bacteria, growth was observed in parallel in media with and without 20 μg mL⁻¹ cycloheximide, 10 μg mL⁻¹ nystatin, and 15 μg mL⁻¹ nalidixic acid added to prevent the growth of yeasts, fungi, other eukaryotic microorganisms, and fast-growing Gram-negative bacteria. The cultures were grown under continuous illumination (10 μmol photon m⁻² s⁻¹) at 25–30°C for about 1–2 months. Then, non-axenic isolated cyanobacteria were grown routinely in both solid and liquid media in continuous illumination of 60–80 μmol photon m⁻² s⁻¹. For some strains, cultures were maintained with rotary shaking (150 rpm) at 30°C and 100 μmol photon m⁻² s⁻¹. Pure cyanobacteria cultures of best-growing isolates were finally obtained by repeatedly streaking on agar-solidified BG11 supplemented with 5% (vol/vol) LB. When needed, light intensity was measured with a PCE-174 luxometer.

An axenic test for those cultures was carried out, as previously described (Taton et al., 2012) with slight modifications. Aliquots of 50 μL of grown isolates were inoculated into 4 different solid media: (1) BG11 with 5% LB (vol/vol), (2) BG11 with 5% LB (vol/vol) and 0.04% glucose (wt/vol), (3) BG11 with 0.01% glucose, 0.01% yeast extract (wt/vol), and 0.01% tryptone (wt/vol), and (4) LB. Plates were incubated at 30 or 37°C in darkness for 1 month. If no growth of heterotrophic bacteria was observed in the plates and by an optical microscope (Leica), the cultures were judged axenic. Axenic isolates were stored at −80°C in BG11 medium supplemented with 5% DMSO (vol/vol).

Synechocystis sp. PCC 6803 and Synechococcus elongatus PCC 7942 were used when needed as a standard cyanobacterial strain. For UV-C studies, E. coli DH10B was used as a reference. For conjugation experiments, E. coli HB101 was used. Cells were grown at 37°C in LB medium, supplemented with 50 μg mL⁻¹ Km and 34 μg mL⁻¹ Cm. Plasmids and strains related to this work are listed in Table 1.

Five milliliters of each isolate were harvested by centrifugation and washed 3 times to reduce the bacteria and extracellular substances present. Then, a rapid DNA extraction with chloroform was made for each cyanobacterial isolate. Single colonies growing on solid media were removed with a sterile plastic tip and resuspended in 100 μL of sterile deionized water. The same volume of chloroform (100 μL) was added to the suspension, vortex for 15 s, and then the mixture was centrifuged at 16.000 g for 15 min at room temperature. 50 μL of the upper, aqueous phase, were transferred to a new tube and used as DNA template for the different PCR applications. The rest of the mixtures were stored at 4°C until use. When bacteria were grown in liquid media, 200 μL culture were centrifugated to obtain a pellet that was washed with sterile deionized water, resuspended in 100 μL water and finally DNA was extracted as previously mentioned.

Polymerase chain reaction (PCR) analysis was carried out using primers for the amplification of the 16S rRNA gene or phycocyanin operon (Supplementary Table S2). PCR amplicons were cleaned with an NZYGelpure kit (Nzytech) according to the manufacturer’s instructions and then directly sequenced at Eurofins Genomics. All the sequence data obtained were analyzed using DNASTAR (Lasergene) programs. Alignments of nucleotide sequences were performed with BLAST.¹

16 rRNA gene sequences from the identified Chroococcidiopsis isolates and the 4–5 most similar sequences were used for phylogenetic analysis. Multiple sequence alignments were performed using MUSCLE algoritm in MEGA-X. Gloeocapsopsis dulcis strain AAB1 (NR_172677.2) was added as an out-group. A neighbor-joining phylogenetic tree was built with the aligned sequences based on the K2 + G + I model with a bootstrap analysis involving 1,000 resampling trees using MEGA-X package.

Axenic isolates of Chroococcidiopsis sp. B11, B13, B14, and B15 were inoculated into 20 mL of BG11 medium in 100 mL Erlenmeyer flasks to an initial optical density at 750 nm (OD_750nm) of 0.05 and grown at different salinity conditions (via NaCl concentration), pH, or nitrogen sources. For temperature experiment tests, 100 mL Erlenmeyer flasks with an initial OD_750nm of 0.20 were used. As a control, isolates grown under routine conditions (BG11 pH 7.5, 30°C, 150 rpm, and continuous light 100 μmol photon m⁻² s⁻¹) were used. Cell growth was monitored by measuring the OD_750nm for a 10-day period. In all the growth experiments, three biological replicates were performed. For Chroococcidiopsis sp. B13 measurements, as it grows in cellular aggregates, 1 mL of culture was first sonicated until aggregates were separated, and then cell integrity was checked by microscopy. To define the relationship between cell density per unit OD at 750 nm wavelength, a hemocytometer was used to count the cells.

To assess growth at different salt concentrations, BG11 medium was prepared containing 0.1, 0.25, 0.5, or 1 M of NaCl. The influence of pH on cyanobacterial growth was explored in BG11 buffered to pH 4, 9, and 11 with 10 mM Tris adjusted to each pH. The temperature effect on growth was evaluated at 4, 40, and 50°C, using 30°C as control. To examine the isolates for growth on different nitrogen sources, BG11 was modified by replacing the 16 mM of NaNO₃ with 16 mM of NH₄Cl or urea. Tolerance to urea was determined by adding this compound to final concentrations of 8 and 16 mM to BG11.

All the strains were inoculated at 0.05 OD_750nm from a 7–10-days old culture and OD measurements were taken every day for 10 days. To calculate growth rates and doubling time, we plotted logOD versus time (hours), and using r = (log [OD2/OD1]) / (T2–T1) in the linear portion. Doubling time corresponds to log2/r. The beginning of the growth phase was considered when the growth of the cyanobacteria was appreciable. One-way Anova with post-hoc Tukey HSD, with Scheffé, Bonferroni and Holm multiple comparison results was calculated for each strain on Astatsa.com (Vasavada, 2016).

To assess the heterotrophic growth of axenic isolates, BG11 agar plates were prepared at final concentrations of 10 mM with different carbon sources: glucose, sucrose, lactose, arabinose, maltose, fructose, galactose, mannose, and glycerol. The tests were performed with spots of 10 μL at OD_750nm = 1 onto BG11 plates to reduce the possibility of contamination. Plates were incubated at 30°C in darkness for 30 days. Furthermore, the photosynthesis inhibitor DCMU [3-(3,4-dichlorophenyl)-1,1-dimethylurea] was added for a final concentration of 10 μM to make sure that the observed growth was heterotrophic. Isolates were checked to ensure that they were free of contaminant bacteria before the experiments.

To determine the total carbohydrate content in the different cultures, phenol-sulfuric acid method was applied as described (Dubois et al., 1956), modified to a microplate format (Masuko et al., 2005). 5 mL samples of 1-month-old culture of each Chroococcidiopsis. isolate, grown without shaking, were used for carbohydrate quantification. This procedure was carried out in the BIOPEN-IMDEA Energy laboratories.

Biofilm formation by Chroococcidiopsis sp. Helios was assayed using crystal violet staining described previously with slight modifications (Allen et al., 2019). Briefly, three milliliters of 2-to 3-week-old Chroococcidiopsis sp. B11, B13, B14, and B15 were cultured in M6 plates at an initial OD_750nm of 0.2, and maintained at 30°C in continuous light (100 μmol photon m⁻² s⁻¹) without shaking for 3 days. As a control, BG11 media without cells were used. After removing the non-adherent cells by aspiration of the medium, cells were washed twice with 2 mL of PBS, and then 0.3% crystal violet in PBS (wt/vol) was added for 10 min to stain the adherent cells. After removing the stain, cells were washed with 2 mL of PBS at least three times. The cells were then suspended in 10 mL of 95% ethanol (vol/vol) for 30 min, and the Abs_588nm was measured. Measurements correspond to cells adhered to the glass. All assays were replicated at least three times.

Cyanobacterial isolates were grown on 9–10 mL of BG11 agar plates (6 cm diameter) under light (60–80 μmol photon m⁻² s⁻¹) at 30°C for 2 weeks with a 30–35% relative humidity in the air. Then, plates were left to be air-dried at under routine growth conditions by removing the parafilm from the Petri dishes. After about 15 days, dried cultures were stored in the laboratory bench room temperature for 3 months, 7 months, and 1 year (on benchtop, RT, 10 μmol photon m⁻² s⁻¹ during the day). For the 1-year dried samples, some samples were maintained in parallel under routine growth conditions. For rehydration, the dried samples were soaked with 1 mL of sterile water for 15 min at room light, streaked on BG11 plates, and incubated under 60–80 μmol photon m⁻² s⁻¹ and 30°C. Results were observed after 2–3 weeks. As a negative control for desiccation tolerance, Synechocystis sp. PCC 6803 was used.

To assess the viability of the isolates to radiation, axenic cultures of cyanobacteria were exposed to different doses of UV-C. Cultures in the late exponential phase of growth were prepared at an OD_750nm of 0.5 in BG11 medium for cyanobacteria, OD₆₀₀ of 0.5 in PBS, for bacteria. We used E. coli and Synechococcus elongatus PCC 7942 as model bacteria for low and moderated UV-C resistance, respectively. Samples without UV-C irradiation were also set as controls. Aliquots of 2 mL were placed in uncovered 6-well cell culture plates (3 cm in diameter) and irradiated with a 460 mW m⁻² UV-C lamp. The irradiation assays were performed in room-light conditions and the doses were adjusted to 250, 500, 750, and 1,000 J m⁻² varying exposure times. The absorbance of BG11 medium at 254 nm was reported to be 0.09 (Tao et al., 2010). Survival after UV-C exposure was tested by colony-forming ability. After irradiation, 1 mL of each sample was collected; then, decimal dilutions were prepared and plated onto agarized BG11 for cyanobacteria and LB for E. coli. Cyanobacterial and bacterial colonies were scored after 2 weeks and 1–2 days of incubation, respectively. The loss of viability was determined by expressing the cell viability as a ratio of the number of viable cells in UV-C-exposed samples to the control (non-exposed) cells calculated as a percentage.

The antibiotic sensitivity of the axenic cultures was evaluated against a set of several antibiotics. Spots of each isolated were plated (10 μL) in BG11 agar with different anbiotics and incubated at 30°C for 7 days. Several concentrations for each antibiotic were tested: 7.5–75 μg mL⁻¹ chloramphenicol (Cm), 20–100 μg mL⁻¹ kanamycin (Km), 25–250 μg mL⁻¹ neomycin (Nm), 2–20 μg mL⁻¹ streptomycin (Sm), 2–20 μg mL⁻¹ spectinomycin (Spt), 2–20 μg mL⁻¹ gentamicin (Gm), 20–200 μg mL⁻¹ erythromycin (Em), and 7.5–75 μg mL⁻¹ nalidixic acid.

A set of cargo-mobilizable plasmids (Table 1) were selected for transfer to the axenic cyanobacterial isolated by spot triparental mating, following published protocols (Elhai and Wolk, 1988). Briefly, conjugation of the different plasmids into all isolates was performed by mixing 0.75 mL overnight cultures of HB101 pRK2013 (conjugative strain) and 1 mL of HB101 cargo plasmid and concentrating the cells in 60 μL. The E. coli mix was incubated for 1 h at 37°C without shaking. On the other hand, cultures 2–3 weeks old of Chroococcidiopsis were centrifuged and the OD_750nm was adjusted from 8 to 0.08 in order to test different ratios of cyanobacteria:bacteria for the mating process. After that, 10 μL was mixed with 10 μL of the cyanobacteria isolate. Mating was carried out on Immobilon-NC filters resting on BG11 agar supplemented with 5% LB for 48 h under conditions used for maintenance growth. Then, filters were transferred to BG11 plates with the appropriate antibiotics. After incubation for 1–2 weeks, isolated transconjugant colonies were patched on fresh selective BG11 plates. The presence of replicative plasmids was confirmed by PCR using specific primers (Supplementary Table S2).

To construct a pSEVA 351-based plasmid, the synthetic strong cyanobacterial promoter P_c223 was used to constitutively express the yfp gene (Markley et al., 2015). The construct was chemically synthetized (Thermofisher) and amplified by PCR using Q5 High-Fidelity DNA Polymerase (New England Biolabs) according to the manufacturer’s protocol. The resulting PCR product was then digested with PmlI y PstI and cloned into pSEVA351 digested with SmaI and PstI. The developed plasmid, named pSEVA351-YFP, was transferred onto Chroococcidiopsis sp. Helios by triparental mating (described above). After plasmid presence confirmation, the transformants were grown on solid BG11 Cm10. After a week, cells were resuspended in 200 μL of liquid BG11 and the OD_750nm was measured. The fluorescence of the different cultures was quantified by transferring 100 μL adjusted to OD_750nm 0.1 in 96-well transparent bottom black plates and measured with a Varioskan reader, using 485/20 and 535/25 nm filters for excitation and emission, respectively. Relative fluorescence was normalized to OD_750nm (Fluorescence/OD_750nm).

Samples collected from solar panels in Valencia were initially plated on BG11, UTEX, MDM, and Castenholz-D medium. In parallel, two of these media were also used without a combined nitrogen source: MDM₀ and BG11₀. No colony could be observed in either of the media lacking a source of nitrogen, but a total of 39 viable colonies were observed in BG11, UTEX, and Castenholz-D. After 2 months of cultivation, only 15 of these viable colonies survived. Single colonies were picked up from each medium, diluted, and streaked again on fresh agar-containing plates. Eight of these isolates were filamentous and seven unicellular, with coccoid form. No fungal contaminations were observed at this step. To eliminate bacterial contamination, isolates with the best growth were re-streaked until no bacteria were detected, as described in MM section 2.1.

Twelve out of fifteen viable colonies were identified by 16S/18S rRNA gene sequence analysis, which revealed that both cyanobacteria (10 isolates) and green algae (two isolates) are present in a solar panel (Table 2). All the best BLAST hits listed in Table 2 are commonly identified in arid locations, where low water availability and high irradiance rates are characteristic (Supplementary Table S3). The 10 cyanobacterial isolates were classified into two orders, including Chroococcidiopsidales and Synechococcales, according to their 16S rRNA sequence (NCBI database). The members in Synechococcales were from various genera including Leptolyngbya, Oculatella, and Myxacorys. Moreover, the green algae Diplosphaera sp. and microalgae Coelastrella sp. (isolates B7 and D14, respectively) were also identified in the sample taken from the solar panel.

Of all the cyanobacterial isolates, only Chroococcidiopsis isolates were unicellular or packet forming, facilitating their handling in the laboratory. The Chroococcidiopsis isolates also had a faster growth rate than other isolates. Taking this into account, we chose the cyanobacterial isolates Chroococcidiopsis sp. B11, B13, B14, and B15 for further characterization (hereafter referred to as Helios isolates or Chroococcidiopsis sp. Helios). The phylogenetic analysis with the Chroococcidiopsis isolates and the 4–5 most similar sequences obtained in BLAST (NCBI database), shows that B13 and B14 are clustering together while B11 and B15 lie in a different branch (Figure 1).

To characterize the growth traits of the chosen isolates, the optical density at 750 nm (OD_750nm) of Chroococcidiopsis sp. B11, B13, B14, and B15 was measured over 10 days in a range of salinity, pH, temperature, carbon, and nitrogen sources. The isolates displayed different growth rates in the conditions assayed (routine maintenance) and consisted of BG11 with pH 7.5, 30°C, 150 rpm, and continuous light (100 μmol photon m⁻² s⁻¹) (Figure 2 and Supplementary Table S4). The relationship between the cell concentration (cell density) and the corresponding optical density at 750 nm (OD_750nm) for the different cyanobacteria was also obtained (Supplementary Table S4). Chroococcidiopsis sp. B15 was the fastest isolate with a doubling time of under 3 days. The other isolates had similar growth rates, being B11the lowest one with a doubling time of 6.6 days. All Helios isolates were pale green, rounded, and tended to be grouped mainly in pairs or tetrads. Chroococcidiopsis sp. B13 had the highest tendency to be aggregated, gathering many tetrads during its growth. It was necessary to sonicate the culture before measurement.

Regarding salt stress, not all the isolates showed the same pattern of response to increasing NaCl concentration (Figure 3 and Supplementary Tables S5–S13). Statistical analysis showed that Chroococcidiopsis sp. B11 growth was clearly improved by the addition of 0.1 M NaCl, since the doubling time was shorter (from 6.7 to 2.8 days) and the maximal absorbance was similar to the control (BG11 medium) (Figure 3 and Supplementary Tables S5, S6). On the other hand, B15 growth was sensitive to NaCl, with doubling time values longer (from 2.4 days of the control to 8.3 days at 0.5 M NaCl) and maximum absorbance values lower than the control in all the concentrations of NaCl tested (Figure 3 and Supplementary Tables S11, S12). B13 was also affected by NaCl presence, mainly by decreasing the maximum absorbance values with the NaCl concentration (Figure 3 and Supplementary Tables S7, S8). Isolate B14 growth was affected mostly at 0.5 M NaCl (Figure 3 and Supplementary Tables S9, S10). No growth was observed at 1 M of NaCl in all Chroococcidiopsis isolates.

The effect of pH on growth was also evaluated and the best pH for growth was the control with 7.5, for all the isolates (Supplementary Figure S1). Chroococcidiopsis sp. B13 was able to grow both at pH 7.5 (control) and 9 while the statistical analysis showed that there was not significant differences between these two conditions (Supplementary Table S7). For Chroococcidiopsis sp. B11, B14, and B15, no growth was detected when the pH was changed, although some cultures did not bleach.

Not only pH but also temperature greatly affected the growth of Chroococcidiopsis sp. (Supplementary Figure S2). None of the isolates were able to grow at 50°C in liquid medium, and the cultures were bleached at the second day of the experiment. According to the statistical analysis the growth was significantly impaired at 4 and 40°C (Supplementary Tables S6, S8, S10, S12). However, at 40°C, after 10 days, the cultures remained green and viable.

Although we initially used nitrate as a source of nitrogen to grow the different Chroococcidiopsis isolates, the effect of its absence or the use of other nitrogen sources for the cell growth was assessed in liquid cultures. All nitrogen sources were prepared at 16 mM, the same concentration as NaNO₃ used in BG11. Figure 4A shows the final OD_750nm after 10 days using alternative nitrogen sources, such as urea or ammonium. None of the isolates were able to grow without any nitrogen source, which may be because they lacked the ability to fix atmospheric nitrogen in the conditions tested. Growth in the presence of ammonium was also closer to the result observed in BG11₀. However, even though all the isolates achieved the maximal absorbance with nitrate, it seems that urea could also be used as a nitrogen source for most of the isolates. In terms of the maximal absorbance only B13 isolate did not presented a significant difference to NaNO₃ under urea concentrations of 16 mM (Figure 4A and Supplementary Table S8). The rest of assayed isolates were affected when growing in 8 mM urea and ammonium (Figure 4A and Supplementary Tables S6, S10, S12).

Considering that urea can be abundant in wastewater, in order to explore the possibility to use these strains in bioremediation, we tested the growth of the different isolates in BG11 with different concentrations of urea (Figure 4B). According to the statistical analysis, the doubling time (9.7 days) was not affected in strains B13 and B14 growing in the presence of urea (Supplementary Tables S7, S9) or even in the case of B11, a shorter doubling time was observed (from the control at 6.7 days to 2.1 days at 16 mM urea), indicating that the presence of urea, probably as an extra source of nitrogen, seems to improve this grow (Supplementary Table S5). By the contrary, the maximal absorbance of isolate B15 decreased in all urea treatments (8 and 16 mM) (Figure 4B and Supplementary Table S12). These results show that even though urea cannot replace NaNO₃ as a nitrogen source, some isolates tolerate it.

Finally, Chroococcidiopsis isolates were tested for heterotrophic growth. We evaluated growth using glycerol and eight different sugars at 10 mM in BG11 agarized medium in complete darkness, and with the photosynthesis inhibitor DCMU at 10 μM final concentration for 30 days. Chroococcidiopsis sp. B11, B13, B14, and B15 were able to grow heterotrophically in the presence of the disaccharides sucrose and maltose but not lactose (Table 3 and Supplementary Figure S3). Regarding monosaccharides, all the isolates tested seem to grow slowly in glucose, while in fructose, only B13 and B14 isolates showed slight growth. Conversely, no growth was detected in mannose, galactose, arabinose, and glycerol for all the isolates tested.

To further evaluate differences among isolates, the percentage of carbohydrate content was determined in the four Chroococcidiopsis isolates (Figure 5A). Isolate B15 with 6.22% had almost half the carbohydrate content of the isolates B11, B13, and B14 (10.32–12.43%). On the other hand, we measured the ability of Chroococcidiopsis isolates to form biofilms (Figure 5B). Here we found that there are some differences among the isolates (Figure 5B). The only isolate that was not able to form biofilms was Chrooccocidiopsis sp. B11, which gave values similar to the control and Synechococcus 7942. The other isolates were able to form biofilms after 3 days without shaking, which is usual in extremophile bacteria (Figure 5B).

Considering that the solar panel could be subjected to periodic absences of water, we investigated the desiccation-tolerance of Chroococcidiopsis isolates. Cultures were plated and subjected to dehydration for 3 months, 7 months, and 1 year, and then rewetted and cultured on solid BG11. Fresh cultures were used as non-desiccated controls. As shown in Figure 6, in terms of ability to form colonies and grow, the rewetted isolates differed little from the non-dried cultivated forms, indicating that all the isolates were drought-resistant. The same result was observed for almost all the isolates from the solar panel (Supplementary Figure S4). Synechocystis sp. PCC 6803, which was used as a non-resistant strain, was unable to grow after 3 months, the minimum time tested (Supplementary Figure S5). On the other hand, the effect of light was negative for dried cultures, as the plates kept in the conditions used for growth maintenance showed blue colonies and did not grow after rewetting (Supplementary Figure S5).

Since the genus Chroococcidiopsis has been already proposed as an ideal candidate for astrobiological applications by several authors (Verseux et al., 2016; Billi et al., 2019; Bothe, 2019), we wanted to present a preliminary approach to UV-C irradiance resistance. Along with the desiccation tolerance, the resistance of Helios isolates to UV-C irradiance was tested. Different doses were used: 250, 500, 750, and 1,000 J m⁻². Survival rates for all the isolates were similar (Figure 7) at above 50%, except for Chroococcidiopsis sp. B14, whose viability dropped below 30%. For comparison, the survival rate of E. coli, used as a UV-C-sensitive control, was around 0.02% at the first dose of UV-C exposure. In contrast, Synechococcus 7942, employed as medium UV-C-resistant control, also survived more than E. coli but far less than Chroococcidiopsis sp. Helios.

In order to have a panel of antibiotics that could be used as selective markers in genetic manipulations, the antibiotic sensitivity of Chroococcidiopsis isolates was evaluated for eight antibiotics in BG11 agar media (Supplementary Table S13). All the isolates were sensitive to chloramphenicol, streptomycin, erythromycin, and neomycin, even at the lowest concentrations checked (25 μg mL⁻¹), but they were resistant to kanamycin, spectinomycin, gentamicin, and nalidixic acid.

To determine if Helios isolates could be transformed after conjugation with E. coli donor cells, a technique that has been used to introduce DNA into a wide variety of cyanobacteria, we performed triparental mating using pRK2013 as a conjugative plasmid, and different cargo plasmids (see Table 1). The chosen plasmids contain origins RSF1010, RK2, pBBR1, and pRO1600/ColE. Additionally, the RSF1010-derived plasmid, which included the CRISPR-Cas12(Cpf1) machinery, was tried. After conjugation, transconjugant colonies of B11, B13, B14, and B15 became apparent on selective mating plates after 1 week (Figure 8A). These isolates showed robust growth after being transferred to fresh selective BG11 Cm (10 μg mL⁻¹) plates and the presence of the plasmid in the transformed isolates was successfully confirmed by PCR (Figure 8B). Thereafter, these results demonstrate that broad host range plasmid vectors based on RSF1010, RK2, pBBR1, and pRO1600/ColE origins can be efficiently transferred to all the Helios isolates (Figure 8A).

To further explore the possibilities of heterologous expression in these new Chroococcidiopsis isolates, we constructed the plasmid pSEVA351-YFP based on the conjugal vector pSEVA351 (Figure 9A). This plasmid contains the YFP under the constitutive synthetic promoter P_c223 (Markley et al., 2015). The pSEVA351-YFP was transferred to Helios isolates by triparental mating. After verifying the presence of the plasmid by PCR (Figure 9B), fluorescence was measured using Chrocococcidiopsis isolates harboring pSEVA351 (empty plasmid) and WT as controls (Figure 9C). The reporter was constitutively expressed in all the isolates, but the result was slightly lower in Chroococcidiopsis sp. B11.