Pseudomonas Vancouvernsis strain DhA-51

The bacteria we found, Pseudomonas Vancouvernsis, is a gram- negative soil bacterium that grows on pulp mill effluents with resin acids. This bacteria has been discovered in the North Western Indian Himalayas where it shows a wide pH range (5–12; optimum 7.0) and salt concentrations up to 5% (w/v) (Mishra, 2008).

References

  •  Mishra, P., Mishra, S., Selvakumar, G., Bisht, S C., Bisht, J K., Kundu, S., Gupta, H S. (2008, September 15). Annals of Microbiology.

Methods for isolation and Identification:

  • There were two particular surfaces that were collected and tested from the Buffalo creek near the Environmental education center ( Figure 1). While collecting the water we tested 100μl of water, 100μl of 1:10 dilution of water, and 100μl of 1:100 dilution of water. For the soil, 0.5 grams of soil was added to 50 ml of sterile water and the same amounts were tested. 
  • A small colony from our undiluted water plate  and a big colony from our 1:100 soil plate were selected for 16S rRNA gene sequencing by PCR amplification ( Figure 2).
  • The product we received was digested and mixed with Msp1; later the product was sent to a sequencing center.Once we received them back, we were able to specify the genus and species of our bacteria.

Figure 1. Collection

IMG950272 IMG950271

Figure 2. Colony selected for identification

Results:

  • Msp1 digestion (figure 3.): A 1,550 bp product was amplified by PCR. With the digestion of Msp1, there really aren’t any bands that are noticeable until it gets closer to the base pair lane.

2017-03-30

Figure 3. Results of PCR amplification and MspI digestion

  • Sequence analysis (figure 4.): The sequenced PCR product generated 812 bases of high-quality reads that were used to identify the genus and species of the colony. The chromatogram of the sequence is available as a pdf (BMB_PREMIX_JF7535_7).   NCBI BLAST analysis showed 96%  identity with bases 28-838 of the 16s rRNA gene of Psuedomonas vancouverensis (Figure 4).

Pseudomonas Van alignment

Figure 4. NCBI BLAST analysis of colony 16-1

Contributed by: Maya Young and Briana Smith, BIOL 250 Spring 2017