Lab 10: Isolation of Antibiotic-producing Bacteria from Soil

BMS 370L - General Microbiology

Dr. J. Mital
12 - 11 - 15
Results:

Gram stain:  Gram-positive

Microscopic characteristics:
Arrangement: strepto- (chains)
Shape: Bacillus (rods)

Colony Morphology:
Table 1: Colony characteristics of the unknown antibiotic-producing bacteria identified as
Bacillus toyonensis
 Characteristic Description

Size Large

Shape Round

Edge/margin Irregular

Chromogenesis Non-pigmented (off-white)

Opacity Opaque

Elevation Flat

Surface Shiny

Consistency Smooth

Zones of clearing for known bacteria:

Table 2: Presence or absence of a zone of inhibition by the antibiotic produced by the
unknown bacteria on plates of known bacteria
Organism Lawn Bacterial unknown #1
Zone of inhibition (y/n)

M. luteus and organism Yes (16mm)

M. luteus and well Yes (1mm)

M. luteus and disc No

S. aureus and organism No

S. aureus and well No

S. aureus and disc No

 S. pyogenes and organism No

S. pyogenes and well No

S. pyogenes and disc No

E. coli and organism No

E. coli and well No

E. coli and disc No

Photographs:

Figure 1: A portion of the chromatogram of the DNA of the unknown soil bacteria,
which is a graphical representation of the sequence used to compare the DNA of the
unknown to other known bacterial DNA and create the phylogenetic tree.
Figure 2: Plates demonstrating the effectiveness of the antibiotic produced by the
unknown soil bacteria against Streptococcus pyogenes and Staphylococcus aureus.
Figure 3: Plates demonstrating the effectiveness of the antibiotic produced by the
unknown soil bacteria against Escherichia coli and Micrococcus luteus.

Figure 4: Phylogenetic tree comparing the 16s rRNA genes of the unknown soil
bacteria and the top three and bottom three BLAST hits.

The unknown antibiotic-producing soil bacteria was most closely related to the

known bacterium Bacillus toyonensis (Figure 4). This, along with the colony morphology

(Table 1) and the microscopic characteristics, suggests that the unknown soil bacteria

tested belong to this species. The effectiveness of the antibiotic produced by this

organism was overall minimal. It was ineffective against three of the four organisms

tested, producing no zones of inhibition whatsoever (Table 2 ; Figure 2 ; Figure 3). A

zone of inhibition of 16mm in diameter was produced on the plate containing M. luteus

around the unknown bacterial colony, and a zone of inhibition of 1mm was produced on

the same plate around the well containing supernatant. No zone of inhibition was

produced around the disk soaked in supernatant (Table 2 ; Figure 3).

Alignments:
Unknown_Soil_Bacteria      
-----------------------GAGCAATGGG-GGGAG---CTATAATGCAAGTCGAGC
Bacillus_toyonensis         ---
GTTTGATCCTGGCTCAGGATGAACGCTGGCGGCGTGCCTAATACATGCAAGTCGAGC
Bacillus_anthracis          
--------------------GATGAACGCTGGCGGCGTGCCTAATACATGCAAGTCGAGC
Bacillus_thuringiensis      ----------
CCTGGCTCAGGATGAACGCTGGCGGCGTGCCTAATACATGCAAGTCGAGC
Bacillus_lentus
AGAGTTTGATCCTGGCTCAGGACGAACGCTGGCGGCGTGCCTAATACATGCAAGTCGAGC
Bacillus_subtilis           
---------------------ACGAACGCTGGCGGCGTGCCTAATACATGCAAGTCGAGC
Bacillus_sonorensis         
----------------------CGAACGCTGGCGGCGTGCCTAATACATGCAAGTCGAGC

Unknown_Soil_Bacteria
GAATGGATTAGAGAGCTTGCTCTCATGAAGTTAGCGGCGGACGGGTGAGTAACACGTGGG
Bacillus_toyonensis         GAATGGAT-
TGAGAGCTTGCTCTCAAGAAGTTAGCGGCGGACGGGTGAGTAACACGTGGG
Bacillus_anthracis          GAATGGAT-
TAAGAGCTTGCTCTTATGAAGTTAGCGGCGGACGGGTGAGTAACACGTGGG
Bacillus_thuringiensis      GAATGGAT-
TGAGAGCTTGCTCTCAAGAAGTTAGCGGCGGACGGGTGAGTAACACGTGGG
Bacillus_lentus             GAATGGAT--GGGAGCTTGCTCCC-
AGAAGTTAGCGGCGGACGGGTGAGTAACACGTGGG
Bacillus_subtilis           GGACAGAT--GGGAGCTTGCTCCC-
TGATGTTAGCGGCGGACGGGTGAGTAACACGTGGG
Bacillus_sonorensis         GAACCGAC--GGGAGCTTGCTCCC-
TTAGGTTAGCGGCGGACGGGTGAGTAACACGTGGG

Unknown_Soil_Bacteria
TAACCTGCCCATAAGACTGGGATAACTCCGGGAAACCGGGGCTAATACCGGATAACATTT
Bacillus_toyonensis
TAACCTGCCCATAAGACTGGGATAACTCCGGGAAACCGGGGCTAATACCGGATAACATTT
Bacillus_anthracis
TAACCTGCCCATAAGACTGGGATAACTCCGGGAAACCGGGGCTAATACCGGATAACATTT
Bacillus_thuringiensis
TAACCTGCCCATAAGACTGGGATAACTCCGGGAAACCGGGGCTAATACCGGATAACATTT
Bacillus_lentus
CAACCTACCTGTAAGACTGGGATAACTTCGGGAAACCGGAGCTAATACCGGATAACTTCT
Bacillus_subtilis
TAACCTGCCTGTAAGACTGGGATAACTCCGGGAAACCGGGGCTAATACCGGATGGTTGTT
Bacillus_sonorensis
TAACCTGCCTGTAAGACTGGGATAACTCCGGGAAACCGGGGCTAATACCGGATGCTTGAT

Unknown_Soil_Bacteria       TGAACTGCATGGTTCGAAATTGAAAGGCGGCT-
TCGGCTGTCACTTATGGATGGACCCGC
Bacillus_toyonensis         TGAACTGCATGGTTCGAAATTGAAAGGCGGCT-
TCGGCTGTCACTTATGGATGGACCCGC
Bacillus_anthracis          TGAACCGCATGGTTCGAAATTGAAAGGCGGCT-
TCGGCTGTCACTTATGGATGGACCCGC
Bacillus_thuringiensis      TGAACTGCATGGTTCGAAATTGAAAGGCGGCT-
TCGGCTGTCACTTATGGATGGACCCGC
Bacillus_lentus             TTCTTCTCCTGGAGAAAGGTTGAAAGACGGCT-
TCGGCTGTCACTTACAGATGGGCCCGC
Bacillus_subtilis           TGAACCGCATGGTTCAAACATAAAAGGTGGCTT-
CGGCTACCACTTACAGATGGACCCGC
Bacillus_sonorensis
TGAACCGCATGGTTCAATTATAAAAGGTGGCTTTTAGCTACCACTTACAGATGGACCCGC

Unknown_Soil_Bacteria
GTCGCATTAGCTAGTTGGTGAGGTAACGGCTCACCAAGGCAACGATGCGTAGCCGACCTG
Bacillus_toyonensis
GTCGCATTAGCTAGTTGGTGAGGTAACGGCTCACCAAGGCAACGATGCGTAGCCGACCTG
Bacillus_anthracis
GTCGCATTAGCTAGTTGGTGAGGTAACGGCTCACCAAGGCAACGATGCGTAGCCGACCTG
 Bacillus_thuringiensis
GTCGCATTAGCTAGTTGGTGAGGTAACGGCTCACCAAGGCAACGATGCGTAGCCGACCTG
Bacillus_lentus
GGCGCATTAGCTAGTTGGTGAGGTAACGGCTCACCAAGGCAACGATGCGTAGCCGACCTG
Bacillus_subtilis
GGCGCATTAGCTAGTTGGTGAGGTAACGGCTCACCAAGGCAACGATGCGTAGCCGACCTG
Bacillus_sonorensis
GGCGCATTAGCTAGTTGGTGAGGTAACGGCTCACCAAGGCGACGATGCGTAGCCGACCTG

Unknown_Soil_Bacteria
AGAGGGTGATCGGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAG
Bacillus_toyonensis
AGAGGGTGATCGGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAG
Bacillus_anthracis
AGAGGGTGATCGGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAG
Bacillus_thuringiensis
AGAGGGTGATCGGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAG
Bacillus_lentus
AGAGGGTGATCGGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAG
Bacillus_subtilis
AGAGGGTGATCGGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAG
Bacillus_sonorensis
AGAGGGTGATCGGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAG

Unknown_Soil_Bacteria
TAGGGAATCTTCCGCAATGGACGAAAGTCTGACGGAGCAACGCCGCGTGAGTGATGAAGG
Bacillus_toyonensis
TAGGGAATCTTCCGCAATGGACGAAAGTCTGACGGAGCAACGCCGCGTGAGTGATGAAGG
Bacillus_anthracis
TAGGGAATCTTCCGCAATGGACGAAAGTCTGACGGAGCAACGCCGCGTGAGTGATGAAGG
Bacillus_thuringiensis
TAGGGAATCTTCCGCAATGGACGAAAGTCTGACGGAGCAACGCCGCGTGAGTGATGAAGG
Bacillus_lentus
TAGGGAATCTTCCGCAATGGACGAAAGTCTGACGGAGCAACGCCGCGTGAGTGATGAAGG
Bacillus_subtilis
TAGGGAATCTTCCGCAATGGACGAAAGTCTGACGGAGCAACGCCGCGTGAGTGATGAAGG
Bacillus_sonorensis
TAGGGAATCTTCCGCAATGGACGAAAGTCTGACGGAGCAACGCCGCGTGAGTGATGAAGG

DNA Sequence of the unknown bacteria:

GAGCAATGGGGGGAGCTATAATGCAAGTCGAGCGAATGGATTAGAGAGCTTGCTCTCATGAAGTTAGCGGCGGACG
GGTG
AGTAACACGTGGGTAACCTGCCCATAAGACTGGGATAACTCCGGGAAACCGGGGCTAATACCGGATAACATTTTGAA
CTG
CATGGTTCGAAATTGAAAGGCGGCTTCGGCTGTCACTTATGGATGGACCCGCGTCGCATTAGCTAGTTGGTGAGGTA
ACG
GCTCACCAAGGCAACGATGCGTAGCCGACCTGAGAGGGTGATCGGCCACACTGGGACTGAGACACGGCCCAGACT
CCTAC
GGGAGGCAGCAGTAGGGAATCTTCCGCAATGGACGAAAGTCTGACGGAGCAACGCCGCGTGAGTGATGAAGGCTTT
CGGG
TCGTAAAACTCTGTTGTTAGGGAAGAACAAGTGCTAGTTGAATAAGCTGGCACCTTGACGGTACCTAACCAGAAAGCC
AC
GGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTATCCGGAATTATTGGGCGTAAAGCGCGC
GCAG
GTGGTTTCTTAAGTCTGATGTGAAAGCCCACGGCTCAACCGTGGAGGGTCATTGGAAACTGGGAGACTTGAGTGCAG
AAG
AGGAAAGTGGAATTCCATGTGTAGCGGTGAAATGCGTAGAGATATGGAGGAACACCAGTGGCGAAGGCGACTTTCT
GGTC
TGTAACTGACACTGAGGCGCGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGAT
GAGT
GCTAAGTGTTAGAGGGTTTCCGCCCTTTAGTGCTGAAGTTTAACGCATTAAGCACTCCGCCTGGGGGAGTACGGCCG
CAA
GGTTGAACTCAAAGGAATTGACGGGGGCCCGCACCAGCGTGGAGCATGTTGGTTTAATTCGAAGCAACGCGAAGAC
CCTT
ACCAGGTCTTGACATCCTCTGAAACCCCTAGATGATAGGCCTTCTCCTTCCGGGAGCAGAGTGAACAGTGGTGCATG
GTT
TGTCGTCAGCTCGTGTCGTGAGATTGTTAGGCTTAAGGTCCGCATCGAGCCGCAATCCTTTGATTCTTAGTTTGACTA
TC
ATTTAATTGGGCACTTCTTAAGTGAACTGGCCGTTGACAACCCGGAGAAGTGGGTATGACTCTATCATCATGCCCCTT
AT
GACTTGCTTACACGTGTCTACATTGACTGATCAAGTAGCTCGTCAGACATCAGCCGAT

Discussion:
Based on both genotypic and phenotypic similarities, the antibiotic-producing
unknown soil bacteria was determined to be Bacillus toyonensis. After preparing and
cleaning the unknown bacterial DNA, specifically the 16s ribosomal RNA gene, it was
sent to a lab for sequencing. The sequence produced by this process is given above.
After BLAST analysis, the three closest matches to the 16s ribosomal RNA gene found
in the unknown soil bacteria were found to be B. toyonensis, Bacillus thuringiensis, and
Bacillus anthracis. The percent identity shows the amount of similarity between the
unknown bacterial DNA and the known bacterial DNA from the database. The
unknown’s percent identity of each of the top three matches was 95%, indicating that
they are all equally close matches. The percent query coverage shows the amount of
overlap between the unknown sequence and the sequence being compared to it. It was
also the same for all three of the top matches, 95%, further demonstrating that they were
all equally genetically similar to the unknown. The gap percentage shows the difference
between lengths of the unknown and known sequences being compared due to
insertions and deletions. When compared to the unknown, each of the top three matches
had the same gap percentage, 2% or 27 gaps. The E-value is used to determine the
reliability of the results; a lower E-value equates to more significant results. The E-value
of the comparisons between the unknown and each of the top three matches all
produced E-values of 0.0, indicating that the other values acquired from the BLAST
search were accurate and reliable. Upon comparison using a multiple sequence
alignment, the unknown bacteria was shown to be closely related to all of the other
bacteria compared, but was not shown to be perfectly matched to any of them (Figure
4). The phylogenetic tree created using this tool was useful as a visual representation of
the genetic relatedness of each of these organisms. It showed that the top three
matches, B. toyonensis, B. thuringiensis, and B. anthracis, were closely related and so
were grouped together in a clade, and the bottom three matches, Bacillus lentus,
Bacillus subtilis, and Bacillus sonorensis, were closely related and grouped into a clade,
but the unknown, while most likely represented by the same genus, was not a part of
either clade. It was, however, shown to be more closely related to the top three matches
and to have a common ancestor with both clades (Figure 4). Since there were three
organisms that were most closely related to the unknown but none that were a perfect
match, this method of identification was useful but inconclusive. After narrowing the
choices down to the most related organisms, microscopic characteristics, colony
morphology, and known information about each species were used to make the final
identification.
After researching each of the three most related organisms, they were compared
to the gram stain and colony characteristics of the unknown soil bacteria, solidifying the
identification of the unknown as B. toyonensis, a gram-positive, rod-shaped species that
often exhibits a strepto- arrangement . This species of Bacillus is typically found in the
2

soil and thrives at temperatures around 35℃ unlike B. thuringiensis and B. anthracis ,
2 5 6
which prefer higher temperatures. B. toyonensis also possesses a colony morphology
that is more similar to the unknown than the other bacteria. The colony characteristics
noted in this experiment may be referenced in Table 1. Unlike the unknown, B.
thuringiensis colonies are not always flat , and B. anthracis colonies are often more grey
5

in color and may be sticky and appear puffy , a characteristic that was not noted for the
6

unknown. Another important factor in the determination of the unknown’s identity was
whether or not the comparable bacteria could produce antibiotics. B. thuringiensis is not
known to produce effective antibiotics against other bacteria, though it does produce
effective insecticides . B. anthracis produces strong exotoxins, but these are far more
4

harmful to eukaryotic host cells than they are to other microbes . B. toyonensis has been
6

shown to possess resistance genes to tetracycline and chloramphenicol, suggesting that

3

it is exposed to antibiotics like these regularly. The production and secretion of an

antibiotic like tetracycline is one of the most likely reasons for the development of these
resistance genes; they would be necessary to avoid hindering of the bacteria’s own
growth by the antibiotic. B. toyonensis is also known to produce and excrete cytotoxic
agents , which are effective against other microbes. The genes encoding these agents
3

are a subject of interest due to the use of this bacteria in Toyocerin feed products .
2

However, they are thought to be dysfunctional due to differences in the amino acid
sequence of the peptides as compared with B. cereus, which produces potent cytotoxic
agents .
1

Of all the organisms tested, the antimicrobial substance secreted by the unknown
was only able to inhibit the growth of M. luteus. It may not have been effective against
the other species studied in the experiment for a number of reasons, including the
development of their own antibiotic resistance genes. On the plate covered with M.
luteus, only the live bacterial colony created a substantial zone of inhibition. The well and
disk full of supernatant were most likely ineffective due to a lack of antibiotic. This could
have been a result of a solution that was too dilute or the fact that bacteria have
mechanisms to suppress their antibiotic-producing genes in nutrient-rich environments
with little competition. Antibiotics are complex molecules and require a lot of energy to
produce; therefore, under optimal conditions such as those created by the broth used in
this experiment, bacteria may refrain from wasting energy on their production. This is
usually accomplished by quorum sensing, a process that utilizes chemical signals for
intercellular communication. When antibiotic-producing bacteria receive these signals
from surrounding, competitive bacteria, they may activate their antibiotic-producing
genes; without these signals, the genes remain inactive and no antibiotic is produced . 7

However, when a live colony of the unknown bacteria was introduced onto the plate
containing other bacteria, it was faced with an array of signals from the other bacteria
and began to actively produce an antimicrobial as it grew.
Overall, this experiment produced sound results. However, the possibility of error
is always present. One possible error associated with experimental procedure was that
the bacteria in the sample used was contained in the tube for a week or two before being
plated in the lab. Taking the bacteria out of their natural environment for too long before
culturing in the lab may have led to the death of some organisms that could have grown
on the TSA plate and produced antibiotics. Another possible error could have occurred
in streaking for isolation. While the bacteria collected seemed to be from a pure and
isolated colony, many different organisms produce large, irregular, flat, nonpigmented
colonies and could easily have been growing on top of one another or near each other
on the same plate. In subsequent experiments, soil may be diluted and plated within a
few days of collection. This would increase the chances of survival for all species living
in the sample. The unknown may be streaked onto multiple plates for isolation, and the
best isolated colonies may then be streaked onto new plates to ensure complete purity.
 In many cases, identification of unknown organisms cannot be based solely on sequential
similarities in DNA, but must be a collaboration of genetic factors and known characteristics.
Molecular and lab techniques as well as current scientific literature on microorganisms are all
equally invaluable in the identification of these organisms, such as the one isolated in this
experiment. Molecular techniques are important in narrowing down the identification to a single
clade within a genus, while supplemental lab techniques such as the gram stain and pure colony
isolation can allow for final identification when compared to current knowledge from scientific
literature regarding typical characteristics, ecology, and antimicrobial capabilities of these microbes.
Using all these techniques, the unknown soil bacteria isolated during this experiment was ultimately
identified as B. toyonensis.

Works Cited:
1. Jiménez et al, (2013) Complete Genome Sequence of Bacillus toyonensis BCT-
7112T, the Active Ingredient of the Feed Additive Preparation Toyocerin.
National Center for Biotechnology Information; U.S. National Library of
Medicine. Web.
2. Jiménez et al, (2014) Description of Bacillus toyonensis sp. nov., a novel
species of the Bacillus cereus group, and pairwise genome comparisons of the
species of the group by means of ANI calculations. Systematic and Applied
Microbiology; (36): 383-391.
3. Aquilina et al. (2014) Scientific Opinion on the safety and efficacy of Toyocerin®
(Bacillus toyonensis) as a feed additive for chickens for fattening, weaned
piglets, pigs for fattening, sows for reproduction, cattle for fattening and calves
for rearing and for rabbits for fattening. European Food Safety Authority (EFSA)
Journal; 12(7): 37-66.
4. Aronson et al, (1986) Bacillus thuringiensis and Related Insect Pathogens.
Microbiological Reviews; 50(1): 1-24.
5. Dorsch et al, (2002) Cry1A Toxins of Bacillus thuringiensis Bind Specifically to a
Region Adjacent to the Membrane-proximal Extracellular Domain of BT-R(1) in
Manduca sexta: Involvement of a Cadherin in the Entomopathogenicity of
Bacillus thuringiensis. Insect Biochemistry Molecular Biology; 32: 1025-36.
6. Friedlander and Heine (2014) Bacillus anthracis (Anthrax). Infectious Disease
and Antimicrobial Agents; Antimicrobe. Web.
Waters and Bassler (2005) Quorum Sensing: Cell-to-Cell Communication in Bacteria.
Annual Review of Cell and Developmental Biology; 21: 319-46.