Difference between revisions of "Team:Paris Bettencourt/Project/Microbiology"

Figure 2 Location of soil samples
collected or obtained by the team.

Preparation of the microbe library

For safety and to avoid environmental contamination, microbial isolation was performed in a fume hood in a BSL 2 facility. More safety information in provided on our safety page.
Microbes were isolated from soil samples using either selective or nonselective plating. For the selective plating, M9 agar was supplemented with either quercetin alone or quercetin with glucose to enrich for microbes with the ability to metabolize quercetin.
The resulting culture was incubated at 30 C for 48 hours, then re-streaked to eliminate potential contamination.
To maximize library diversity, we preferentially chose colonies with unique morphologies. After isolation, we performed colony PCR with universal 16s rRNA primers (see methods). Sequencing the resulting PCR products allowed us to identify the strains and position them within the greater bacterial taxonomy.

Quercetin degradation assay

Each of the 189 isolated strains was innoculated into M9 quercitin medium in triplicate and incubated at 37 C for 6 days. At the end of the period, quercitin concentration was measured by absorbance.

Of 186 strains tested, 50 produced quercetin levels significantly lower than controls (Figures 3 and 4). 20 strains degraded more than 50% of quercetin and 2 strains degraded more than 80%.
Both selective and nonselective plating methods were able to produce quercetin-degrading strains. 4 of the 5 strains showing the most quercetin degradation were obtained by selective plating.
However, 40 of the 50 strains showing significant degradation were obtained by nonselective plating.

Figure 3 Quercetin degradation by 186 microbes collected from global soil samples. Single colonies were inoculated in M9 minimal medium and grown for 6 days. Quercetin was measured by absorbance. Strains to the extreme left of the figure represent the highest-degrading strains. Red bars represent strains isolated from selective media and blue from non selective media.

Figure 4 Quercetin degradation detail. The top-performing strains included those isolated from both selective and nonselective media. Strains marked with an asterisk were selected for further investigation. Red bars represent strains isolated from selective media and blue from non selective media.

Working with M9 quercetin plates can be challenging. The bright green color of the plates makes colony visualization difficult.
Indeed, only fungal mycelia were visible due to a white halo resulting from quercetin degradation. However, working with liquid media allows the control of residual sample carbon source contamination through sample dilution.
Additionally, we were concerned agar in plates could be used as a carbon source.

8 top-performing strains were selected for detailed valication in a time-course assay, and all were able to degrade quercitin significantly better than E. coli negative controls.
We observed a 50% degradation over 6 days in the best case.

Figure 5 Kinetics of quercetin degradation by 8 promising strains. This experiment had two negative controls, one with no bacteria (black line) and one with non-quercetin degrading E. coli. Pseudomonas putita was included as a positive control. Four strains, shown in bold, degraded quercetin at a higher rate than our P. putida control.

Phylogenetic analysis of quercetin degradation

Using the 16s rRNA sequences, we constructed a global phylogeny of all the assayed strains (Figure 6).
Quercitin-degrading activity was concentrated among the Pseudomonas genus, where most of the strains were positive.
However many diverse species were able to significantly degrade quercitin, suggesting that the phenotype may be highly variable in evolution.

Figure 6: Phylogenetic Tree of isolated strains. We constructed a phylogenetic tree of all isolated bacterial strains. Strain taxonomic classification is indicated by the color key to the left of the figure. Strains that demonstrated high quercetin degradation are marked with an asterisk. Those strains marked with a large star were selected for whole genome sequencing to look for common anthocyanin-degrading genes.

Mining genomes for quercetin-degrading enzymes

Four of our top-degrading strains, representing diverse phylogentic lineages, were selected for whole genome sequencing.
As of the wiki freeze, the genome sequences were not complete. So, as a proxy, we obtained complete genome sequences of their nearest relatives in GenBank.
BLAST searches against the genomes revealed the presence of several candidate quercitin-degrading enzymes, including laccase and XylE.

The results of these genomic analysis were passed to the enzyme group, where these enzymes form the basis of our synthetic stain fighting product.

Figure 7: Genome table. Genome sequences were analyzed for the presence of potential anthocyanin degrading enzymes as identified in the Enzymes Project. The genes of at least one of the six candidates were identified for each of the sequenced strains. The number of + signs indicated represent the number of gene copies in the bacterial genome.

Methods

Anthocyanin extraction

Vinis vitifera grapes were our source for anthocyanin extraction.
Grapes were peeled and the skins were collected, washed and soaked overnight in ethanol with 1% HCl.
By trial and error, we determined that 2.5 mL of this solution per 1 g of skins was the best compromise between efficiency and the quantity of solvent used.
The solution was filtered with Whatman paper, with the filtrate collected, and the solvent evaporated at 37°C for several hours.
The dry extract was resuspended in water (10 mL for 1g of grape skin).

Anthocyanin quantification by differential absorbance

Following Lee et al. (2005), we prepared one buffer at pH 1 (0.025M potassium chloride) and a second at pH 4.5 (sodium acetate, 0.4M).
100 µL of the anthocyanin solution was mixed with 900µL of each buffers and the color was allowed to develop over 20 minutes.
Absorbance measurements were obtained at 510 nm and 700 nm for each solution.
Anthocyanin concentration was determined as a function of the four absorbance measurements, using an established formula (Lee et al., 2005).

Protein quantification with Bradford assays

A stock solution of Bovine Serum Albumin (BSA) was prepared in water at 1 mg/mL.
100 μL of standard dilutions of BSA solution were mixed with 1 mL of Bradford Reagent and mixed by vortexing.
Absorbance was measured at 595 nm. Experimental samples were treated similarly and compared to the BSA standard curve to determine concentration.

Carbohydrate quantification with Fehling Reaction

200µL of Fehling's A solution, 200µL of Fehling's solution B and 200µL of our carbohydrate solution into sodium acetate buffer (20µL of solution and 180 µL of buffer).
The Fehling reaction is measured as the loss of absorbance at 650nm relative to a blank solution without carbohydrate.
Quantification was achieved by comparison to a standard curve of glucose prepared at 1g/L to 5g/L.

Bacteria plating on selective and non-selective media

1 g of soil samples were suspended in 5 mL Phosphate Buffered Saline (PBS) then left to stand allowing large particles to settle.
The soil suspension was serially diluted to obtain a suitable density of microbes (typically 1:1000) then 200 µL was plated on standard Petri dishes with M9 agar with 1 g/L quercetin for selection.
Non-selective plating was performed on a range of rich media including FTO agar (Curry, 1976), Mossel agar (Mossel, 1967), standard LB, standard TSA and standard M9 glucose.

Protocol for growth assay in Quercetin M9 liquid media

Following the protocol of Dantas et al. 2012, all step were performed in liquid media to control soil carbon source contamination.
We suspended soil samples in 5 ml M9 with 1g/L quercetin at pH 7 in 50 ml Falcon tubes with 500µL of overnight culture of strains isolated from selective or non-selective plates.

All cultures were made in triplicate at 30°C with shaking at 150 rpm for several days. As quercetin is not soluble at pH=7, shaking important to avoid precipitation.

Quercetin absorbance measurement

Quercetin absorbance was measured at two time points for histogram construction: at 0 days to ensure quercetin sample concentration consistent with the controls, and at 6 days to evaluate quercetin degradation.
M9-quercetin and Pseudomonas putidaK2440 samples were included as negative and positive controls, respectively.

Prior to absorbance measurement, Quercetin was solubilized by diluting samples 10 fold in 0.5M NaOH, centrifuged to remove cell material, and further diluted 100X for measurement at 315 nm in a Tecan plate reader.

PCR for 16s characterization, sequencing interpretation and phylogenetic tree construction.

To identify bacterial strains, 16S rRNA sequences were amplified through colony PCR, column purified, and Sanger sequenced by GATC.
The resulting sequences were submitted for BLAST comparison at ncbi.gov.
Alignments were performed using the Ribosomal Database Project Aligner tool (https://rdp.cme.msu.edu/),
and a phylogenetic tree was constructed using Geneious software with the following parameters: we used Neighbor-Joining tree building with Jukes Cantor as the genetic distance model, with a 93% similarity cost matrix for the alignment with free end gaps.
The tree was then exported and improved using the online Tree of Life software (http://www.tolweb.org/tree/).

PCR for genome sequencing.

We isolated bacterial DNA using the DNeasy Blood and Tissue Kit from Qiagen.
We submitted four strains to GATC for whole genome sequencing: NS.4 (Lysinibacillus), S.48 (Stenothrophomonas maltophilia), S.33 (Oerskovia Paurometabola), NS.33 (Microccocus Luteus) according to their sample preparation specifications.

Attributions

This project was done by Antoine Villa Antoine Poirot and Sébastien Gaultier. Anthocyanin data was obtained by Ibrahim Haouchine.
Thanks to our advisors Jake and Jason for all their help with the figures.
We would like to thank Philippe Morand from the microbiology lab of Cochin for his advice.

References

• Kanekar, P. P., Sarnaik, S. S., & Kelkar, A. S. (1998). Bioremediation of phenol by alkaliphilic bacteria isolated from alkaline lake of Lonar, India. Journal of applied microbiology, 85(S1).
• Dantas, G., Sommer, M. O., Oluwasegun, R. D., & Church, G. M. (2008). Bacteria subsisting on antibiotics. Science, 320(5872), 100-103.
• Lee, J., Durst, R. W., & Wrolstad, R. E. (2005). Determination of total monomeric anthocyanin pigment content of fruit juices, beverages, natural colorants, and wines by the pH differential method: collaborative study. Journal of AOAC international, 88(5), 1269-1278.
• Curry, J. C., & Borovian, G. E. (1976). Selective medium for distinguishing micrococci from staphylococci in the clinical laboratory. Journal of clinical microbiology, 4(5), 455.
• Pillai, B. V., & Swarup, S. (2002). Elucidation of the flavonoid catabolism pathway in Pseudomonas putida PML2 by comparative metabolic profiling. Applied and environmental microbiology, 68(1), 143-151.
• Herrmann, H., Janke, D., Krejsa, S., & Kunze, I. (1987). Involvement of the plasmid pPGH1 in the phenol degradation of Pseudomonas putida strain H. FEMS microbiology letters, 43(2), 133-137.

---

Assay

---

Microbiology

---

Enzyme

---

Binding

---

Indigo

---

Model