Environmental strains results | UppsalaUniversity

Subproject Achievements

Successes

Discovered two bacterial pigments that most likely are chromoproteins
Developed a Chromosearch Pipeline to easily identify candidate chromoprotein or pigment synthetizing enzymes from genome data

Failures

Were unable to identify the chromoproteins we found

Lysis from Liquid

Following the lysis process, three environmental strains (91, 153, and 350) exhibited colored supernatants. This indicated the potential presence of chromoproteins or pigment-associated particles in these strains (Figure 1, 2 and 3).

**Figure 1:** Supernatant and pellet of strain 91 after lysis

**Figure 2:** Supernatant and pellet of strain 153 after lysis

**Figure 3:** Supernatant and pellet of strain 350 after lysis

Size Exclusion Chromatography

We performed size exclusion chromatography (SEC) on the lysates from strains 91 and 350, alongside the known chromoprotein mRFP1, using Cytiva’s PD-10 desalting columns packed with Sephadex G-25 resin (5 kDa cut-off). We also performed this SEC with bromophenol blue (a non-protein small molecule). When the bromophenol was run alongside strain 350, a clear separation was observed (Figure 4). This supports the hypothesis that strain 350 was either a protein or a molecule bound to a protein.

In contrast, when mRFP1 was run alongside strain 91, no clear separation between mRFP1 and the chromophore could be observed, suggesting that the chromophore in strain 91 was approximately the same size as mRFP1 (Figure 5). Similarly, strain 350 produced comparable results, indicating a protein-like chromophore.

Figure 4: SEC run with bromophenol blue and strain 350

Figure 5: SEC run with mRFP1 and strain 91

Concentration

conc_1 — **Figure 6:** Eluted samples from strain 350 and 91 before being concentrated using spin columns.

Figure 7: Eluted samples from strain 350 and 91 after concentration via spin columns, showing the final volume reduction

After SEC, the chromophores in the samples were diluted in the elution buffer, resulting in fainter colors (Figure 6). To concentrate these samples, we used GE Healthcare Amicon Ultra-15 3,000 MWCO protein concentrator spin columns with a 3 kDa molecular weight cut-off. Post-concentration, the sample volumes were reduced, and the colors intensified (Figure 7), allowing for better visualization in downstream analysis.

Semi native SDS-PAGE run

Figure 8: Semi-native SDS-PAGE gel showing protein ladder and samples from strains 91, 350, and mRFP1 chromoprotein

This gel shows the results of our run, with lanes 2-7 containing samples from our environmental strains 91 (lanes 2, 4, 6) and 350 (lanes 3, 5, 7). Both strains exhibit a faint yellow, broad band around the 25 kDa marker. Lanes 8 and 9 contain the control chromoprotein, mRFP1. To avoid interference from the color of the bromophenol blue in the loading dye, we adjusted its concentration across different lanes. Specifically, we used 90% of the original dye concentration in lanes 10, 5, and 4, 60% in lanes 6 and 7, and 20% in lanes 2, 3, and 9. For the control protein mRFP1, we used both 20% (lane 9) and 90% (lane 8) dye concentrations.

Figure 9: Semi-native SDS-PAGE gel of Figure 8 but with enhanced yellow hue for better visualization of bands.

Concentrated samples from strains 91 and 350 were analyzed via semi-native SDS-PAGE. In lanes 2–7, yellow bands were observed at around 25 kDa (Figure 8), suggesting the presence of potential chromoproteins. For enhanced visibility, the yellow hues in the gel were digitally adjusted, clearly highlighting the broad bands that may correspond to the chromoproteins of interest (Figure 9).

Discussion

Our results indicate that two of the investigated environmental strains (91 and 350) have the potential to produce chromoproteins or other protein-associated dyes that could be used as bio-dyes. This is supported not only by the SEC experiments but also by bioinformatic analysis, which identified genes in these strains associated with chromophore production. The bioinformatic predictions from our Chromosearch pipeline aligned with the SDS-PAGE findings, strengthening the hypothesis of chromoprotein presence in these strains. The lack of separation between these dyes and mRFP1 indicates that the dye components have similar sizes and are thus likely to be protein-bound.

Strain 153, which initially showed color in the supernatant, was removed after the SEC experiments due to poor separation and the presence of mucus, which interfered with column function. This may suggest that some environmental strains contain components that can complicate protein purification, which is important to consider in future experimental protocols.

Through the use of protein concentrator spin columns, we were able to successfully concentrate the eluted sample volumes, allowing the detection of chromoproteins on the SDS gel. On the semi-native SDS gel, a faint yellow band was observed at around 25 kDa, suggesting that we may have identified a novel chromoprotein. However, as semi-native conditions were used, the size estimate is only approximate, and further purification and analysis is required to confirm these results.

It is also worth noting that the use of both water- and non-water-soluble solvents for pigment extraction from pellets may be an important step forward in our understanding of non-water-soluble chromophores. Several strains, especially strain 91, showed color in the pellets after lysis, indicating that the dyes may be membrane-bound or of a more hydrophobic nature.

In conclusion, the hypothesis that our investigated environmental strains can produce chromoproteins or chromophores that are interesting for further investigation and could potentially be used as bio-colourants is supported. However, further work remains to be done to fully identify and characterize these proteins.

Conclusion

Two potential environmental strains (91 and 350) have been identified that exhibit indications of chromoprotein production. Through a combination of cell lysis, SEC, protein concentration and semi-native SDS-PAGE, we were able to demonstrate the presence of a color component at approximately 25 kDa, which may be a chromoprotein. In addition to experimental evidence, bioinformatic analysis supported these findings by identifying chromophore-related genes in both strains, further reinforcing their potential for producing bio-colourants. Despite these promising results, further research is required to ensure the identity, purity, and potential of the proteins for use as bio-colourants. This study represents an important first step towards discovering and developing sustainable alternatives to synthetic dyes for the textile industry.