Vickie Nguyen and Haylie Beall

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Classification

Bacillus subtilis

Domian: Bacteria

Kingdom: Monera

Phylum: Firmicutes

Class: Bacili

Order: Bacillales

Family: Bacillaceae

Genus: Bacillus

Species: B. subtilis

NCBI: Taxonomy

Laboratory and Site Collection Data

The organism was isolated from a 10^-3 dilution of soil harvested from a patch of soil in Buda, TX. The soil sample was collected on a sunny afternoon in February 2016. The weather showed low humidity and a temperature of 83 degrees Fahrenheit. The soil dilution was plated on a TSA plate and cultured for one week. Bacterial colonies were transferred to a master patch plate and tested for antibiotic resistance. The organism was chosen based on its antibiotic resistance to S. aureus.

According to BLAST using a genome sequence from PCR, our organism has been classified as Bacillus subtilis. B. subtilis is one of the most widely researched and most commonly found bacterium used in research. It has been studied to gain insight into bacterial transformation, bacterial growth, and biochemical properties. Today vaccine research is studying B. subtilis in efforts to incorporate the bacterium into a new drug mechanism

History of Bacillus subtitles

Bacillus subtitles was discovered by the nazi German medical corps in 1941. At the time, the German military was its height in both Africe but alarm set in when hundreds of soldiers began dying every week from dysentery. The Germans were aware that dysentery was caused by pathogenic bacteria from the local water sources, but with no antibiotics the nazis quickly began looking for other means to help their dying soldiers. They sent out scientists, physicians, and chemists to help find the solution. They came to the conclusion that the people currently living in North Africa must have been exposed to the bacteria but that they had a "cure" for the sickness. They watched to see why they were unaffected by the pathogen and they discovered that once one of the native people started showing signs of dysentery, the people would follow around a horse/camel until it would drop its dung and then they would quickly ingest the droppings. This would effectively eliminate the dysentery almost overnight. Once the Nazia witnessed this they began examining fresh camel and horse dung discovering that it was teeming with a powerful microorganism that later became identified as Bacillus subtitles. The bacteria it turned out was so strong that it practically cannibalized the virulent strain which was causing dysentery.

Habitat Information

Bacillus Subtilis is typically found in upper layers of soil but is present in the air, water and plant compost. " The soil bacterium can divide asymmetrically producing an endospore that is resistant to environmental factors such as heat, acid and salt, and can persist in the environment for long period of time. The endospore is formed at times of nutritional stress, allowing the organism to persist in the environment until conditions are favorable,"[1]

Description and Significance

The organism has a colony morphology that is large(up to 2cm), white, rough(blistery with veins), and has irregular borders. There was a clearing surrounding the colony on a test patch plate containing E. coli. We have concluded that it has possible antimicrobial properties against E.coli. Under the microscope, we identified the organism to be gram positive with endospores using gram staining and endospore staining procedures. The organism were arranged in singular rods without a capsule. Because B. subtilis can be found readily in the soil and near grass, its usage of antimicrobial properties may be both practical and effective against S. aureus infections.

Bacillus subtilis is useful in many ways, including industrial applications . Bacillus subtills is also used to produce enzymes including amylase and protease. It is also used in used to produce a variety of antibiotics such as difficidin and oxydifficidin.

Genome Structure

B. subtilis is thought to have a genome containing ~4100 genes. It replicates its genome through natural bacterial transformation where a competent bacterium transfers its double stranded circular DNA to an incompetent bacterium. Below are the forward and reverse sequences obtained from PCR.

Forward: ACGGAGCAACGCCGCGTGAGTGATGAAGG TTTTCGGATCGTAAAGCTCTGTTGTTAGGGAAGAACAAGTACCGTTCGAATAGGGCGGTACCTTGACGGTACCTAACCAG AAAGCCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTGTCCGGAATTATTGGGCGTAAAGG GCTCGCAGGCGGTTCCTTAAGTCTGATGTGAAAGCCCCCGGCTCAACCGGGGAGGGTCATTGGAAACTGGGGAACTTGAG TGCAGAAGAGGAGAGTGGAATTCCACGTGTAGCGGTGAAATGCGTAGAGATGTGGAGGAACACCAGTGGCGAAGGCGACT CTCTGGTCTGTAACTGACGCTGAGGAGCGAAAGCGTGGGGAGCGAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAA CGATGAGTGCTAAGTGTTAGGGGGTTTCCGCCCCTTAGTGCTGCAGCTAACGCATTAAGCACTCCGCCTGGGGAGTACGG TCGCAAGACTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCAACGCGAA GAACCTTACCAGGTCTTGACATCCTCTGACAATCCTAGAGATAGGACGTCCCCTTCGGGGGCAGAGTGACAGGTGGTGCA TGGTTGTCGTCAGCTCGTGNCGTGA

Reverse: ACCACCTGTCACTCTGCCCCCGAAGGGGACGTCCTATCTCTAGGATTGTCAGAGGATGTCAAGACCTG GTAAGGTTCTTCGCGTTGCTTCGAATTAAACCACATGCTCCACCGCTTGTGCGGGCCCCCGTCAATTCCTTTGAGTTTCA GTCTTGCGACCGTACTCCCCAGGCGGAGTGCTTAATGCGTTAGCTGCAGCACTAAGGGGCGGAAACCCCCTAACACTTAG CACTCATCGTTTACGGCGTGGACTACCAGGGTATCTAATCCTGTTCGCTCCCCACGCTTTCGCTCCTCAGCGTCAGTTAC AGACCAGAGAGTCGCCTTCGCCACTGGTGTTCCTCCACATCTCTACGCATTTCACCGCTACACGTGGAATTCCACTCTCC TCTTCTGCACTCAAGTTCCCCAGTTTCCAATGACCCTCCCCGGTTGAGCCGGGGGCTTTCACATCAGACTTAAGGAACCG CCTGCGAGCCCTTTACGCCCAATAATTCCGGACAACGCTTGCCACCTACGTATTACCGCGGCTGCTGGCACGTAGTTAGC CGTGGCTTTCTGGTTAGGTACCGTCAAGGTACCGCCCTATTCGAACGGTACTTGTTCTTCCCTAACAACAGAGCTTTACG ATCCGAAAACCTTCATCACTCACGCGGCGTTGCTCCGTCAGACTTTCGTCCATTGCGGAAGA

Cell Structure, Metabolism and Life Cycle

Under the microscope, we identified the organism to be gram positive with endospores using gram staining and endospore staining procedures. The organism were arranged in singular rods without a capsule. B. subtilis can be found readily in the soil and near grass, and we suspect thatit will have a long life cycle due to resilient heat resistant spores to carry on its genetic material. That being said, B. subtilis can also reproduce via binary fission.

Physiology and Pathogenesis

According to the biochemical reactions listed below, our organism is motile with the capabilities of fermenting Glucose and Sucrose. It causes a neutral reaction in the MRVP test and is capable of lipid hydrolysis. It is also positive for citrate and nitrate reduction. Other biochemical tests, listed below, show that the organism is gram (+), salt tolerant, and not capable of lactose fermentation. The decarboxylation test shows that it had (+) fermentation for Arginine, Ornithine, and Lysine. In blood agar, it is capable of partial hemolysis, and is also (+) for esculin hydrolysis. Subjection to disinfectants revealed that it is sensitive to Lysol and clove but resistant to isopropyl alcohol and bleach. Fortunately, B. subtilis is not known to cause disease.
Motility: yes
Phenol Red Broth:
Glucose: Yellow, no gas, (+) fermentation
Lactose: Pink, no gas, (-) fermentation
Sucrose: Yellow, no gas, (+) fermentation
Starch Hydrolysis: iodine turns black, (-)
Casein Hydrolysis: skim milk agar, no clearing, (-)
DNA Hydrolysis: (-)
Lipid Hydrolysis (Tributyrin): clearing in agar, (+)
Methyl Red: unchanged (-)
Voges Proskauer: red (+)
Citrate Test: blue/green (+)
SIM Tests: (-) motility, (-) indole
Nitrate Reduction: Red after adding Reagents A + B, (+) nitrate to nitrite
Urea Hydrolysis: no change, (-) Urea hydrolysis
Triple Sugar Iron: orange/pink, (-) fermentation of lactose, sucrose, and glucose
Oxidase: no change, (-)
EMB Agar: no growth, Gram (+), fermentation (-)
Hektoen Enteric Agar: no growth, Gram (+), lactose fermentation (-)
MacConkey Agar: no growth, Gram (+), lactose fermentation (-)
Decarboxylation: yellow, (-) decarboxylation and (+) fermentation for Arginine, Lysine, and Ornithine
Phenylalanine Deaminase : no change (-) fermentation
Catalase: no bubbles (-)
Blood Agar: green clearing, partial hemolysis
Mannitol Salt Agar: pink, growth, (-) mannitol fermenation, salt tolerant gram (+)
Phenylethyl Alcohol: growth, gram (+)
Salt broth: fine uniform turbidity, (+) salt tolerance
No growth on the Bacitracin/Optichin blood agar plate
Bile esculin: dark brown, (+) esculin hydrolysis and salt tolerance
Disinfectant Clove: sensitive
Disinfectant 10% Lysol: sensitive
Disinfectant 100% Lysol: sensitive
Disinfectant Bleach: resistant
Disinfectant Isopropyl Alcohol: resistant

References

[1] -Amuguni, Hellen, and Saul Tzipori. “Bacillus Subtilis: A Temperature Resistant and Needle Free Delivery System of Immunogens.” Human Vaccines & Immunotherapeutics 8.7 (2012): 979–986. PMC. Web.
[2] - Bauman, Robert W. Microbiology: With Diseases by Body System. Print.
[3] - Leboffe, Michael J., and Burton E. Pierce. Microbiology: Laboratory Theory & Application. Print.
[4] - National Center for Biotechnology Information."Summary." U.S. National Library of Medicine. Web.

Author

Page authored by Vickie Nguyen and Haylie Beall, students of Prof. Kristine Hollingsworth at Austin Community College.