Bacillus toyonensis is a Gram-positive motile bacteria that was observed twice prior to this study by Jiménez and colleagues (2013A, 2013B) on plated cultures. The cultured bacteria grew flat, granulated, and creamy white colonies.

References:

•  Jiménez G, Urdiain M, Cifuentes A, López-López A, Blanch A, Tamames J, Kämpfer P, Kolstø A, Ramón D, Martínez J. 2013A. Descriptionof Bacillus toyonensis sp. nov., a novel species of the Bacillus cereus group, and a pairwise genome comparisons of the species of the group by means of ANI calculations. Elsevier. 36(6):383-391.
•  Jiménez G,  Blanch A,  Tamames J ,  Rosselló-Mora R. 2013B. Complete Genome Sequence of Bacillus toyonensis BCT-7112, the Active Ingredient of the Feed Additive Preparation Toyocerin. Genome Announcements. 1:6-13.

Date Collected: February 9, 2017

Methods for isolation and identification:

• Five milligrams of  soil was collected from the runoff basin located at Lancer Park (Figure 1). This sample was mixed with twenty-five milliliters of sterile water , then plated and spread with a sterile swab. The plate was then incubated for several hours.
•  After incubation, a single white bacteria colony was chosen from the direct count runoff basin agar plate (Figure 2). DNA extraction and PCR purification was performed using the colony.
• The PCR product was digested by the use of MspI and sent off for sequencing. BLAST was used to identify the bacteria species from the sequencing results.

 

Figure 1. The Runoff Basin located at Lancer Park

 

Figure 2. Colony selected from the Runoff Basin plate

Results:

• MspI Digestion (Figure 3): A 1032 base pair product was amplified by PCR. The PCR was digested using a MspI, four bands can be seen on the gel at approximately  at bp 600, bp 400, bp 200, and bp 150.

Figure 3. Results of 12R PCR amplification and MspI digestion 

• Sequence analysis (Figure 4): The 12R PCR product generated 655 bases of high quality reads. These bases were used in a program called BLAST, to identify the bacteria colony’s genus, species, and strain. The chromatogram sequence can be viewed  as PDF  in (12R_PREMIX_JF7521_16)
• The NCBI BLAST showed a 99% similarity with the 16s rRNA gene of Bacillus toyonensis strain BCT-7112 complete sequence, with a similarity of bases 1-511 (Figure 4).

 

Figure 4. NCBI BLAST analysis of Colony 12R

Contributed by: Erica Harris, Morgan Karnes, Bio 250 Spring 2017, Group 12