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Identification b bacterial genes known to be involved in antibiotic synthesis pathways

By Mirvat Kalouch, M.S.

A Thesis Submitted to the Department of Biology California State University Bakersfield In

Partial Fulfillment of the Degree of Masters of Biology

Summer 2017

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Copyright

By

Mirvat Kalouch

2017

Identification of bacterial genes known to be Involved in antibiotic synthesis pathways

By Mlrvat Kalouch

This Thesis of project has been accepted on behalf of the Department of Biology by their supervisory committee:

Nam~~~~\hl ~eA

Name of Committee Member f:\ m be(' S\o 14 e S

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Table of Contents

Introduction

Amphibians 2

Infectious diseases 2

Chytridiomycosis 2-3

Antibiotics 3

Methods

Results

Antibiotics produced by Bacillus 3-12

Cultivation of bacterial isolates 13

DNA extractions and PCR 13

PCR product purification and sequencing 14-16

Confirmation of bacterial isolate species 17

Confirmation of positive control and PCR analysis 17-45

Discussion 46-51

Significance 52-53

Conclusion and future projects 53-54

Literature Cited 55-59

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Identification of bacterial genes known to be involved in antibiotic synthesis pathways

Introduction

Amphibians

Over the past 25 year’s numbers and populations of amphibians worldwide are declining

dramatically due to factors such as habitat destruction, overuse of pesticides, and re-emergence of

known or previously established diseases (Beebee and Griffith 2005). Development and

construction also disrupts natural habitat and causes a decrease in population sizes. Also, the

overuse of pesticides kills more than just insects resulting in a decrease in amphibian numbers

(Beebee and Griffith 2005). Infectious diseases are particularly threatening to wildlife species

including amphibians (Daszak et al. 1999).

Infectious diseases

In humans, infectious diseases are one of the main causes of death in the United States and

are believed to increase in the near future (Spellberg et al. 2008). One fifth of global deaths are due

to infectious diseases including respiratory tract infections, tuberculosis, malaria, and diarrheal

diseases. This increase is due to the fact that infectious agents are evolving at the same rate, if not a

faster rate, as humans (Lederberg et al. 1992). With modern transportation, pathogens are able to

spread across the world because they are constantly changing on a genetic level and are able to

target and successfully infect new hosts (Lederberg et al. 1992). Infectious diseases can be spread

through insects such as mosquitoes, animal bites, contact with soil or water, or by human to human

contact (SA Health). Furthermore, insects are becoming increasingly resistant to insecticides;

therefore, increasing the rate of transmission of infectious diseases that they spread (Lederberg et

al. 1992). The environment also plays a role in that it is constantly changing due to urbanization

and industrialization, which allow for the emergence of new pathogens and re-emergence of known

pathogens (Lederberg et al. 1992). These changes in the environment allow pathogens to adapt to

and better survive new changes.

Chytridiomycosis

One disease attributing to the decline of amphibians in the Americas, Europe, Australia, and

New Zealand is chytridiomycosis, caused by the fungal pathogen Batrachochytrium dendrobatidis

(Bd) (Beebee and Griffith 2005). This disease attacks the skin of amphibians impairing

osmoregulation and respiration, resulting in death (Beebee and Griffith 2005). As mentioned above

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amphibian populations around the world have been impacted by environmental changes, invasive

species, climate change, and pathogens (Daszak et al. 1999). The North American bullfrog

(Lithobates catesbeiana) and California toad (Anaxyrus boreas halophilus) are surviving in

environments that are similar to those where other frog and toad populations are declining (Reeder

et al. 2012). Survival may be due to cutaneous bacteria on amphibian skin that produce antibiotics

protecting individuals from infection (Austin 2000 and Szick et al. 201X).

Antibiotics

Antibiotics are used to treat infections caused by bacteria and work by causing cell death or

preventing cell growth and, in turn, helping the body’s immune system fight off the disease.

Antibiotics are used as bacteriostatic drugs and bactericidal drugs, which are two categories of

antimicrobial therapies. Bacteriostatic drugs inhibit ribosome function by targeting the 30S and 50S

subunits; the 30S subunit includes the aminoglycoside family of drugs, which is known to cause

protein mistranslation and have an efficiency of about 99.99% (Kohanski et al. 2007). In order to

determine if an antibacterial agent is bactericidal or bacteriostatic, growth conditions, bacterial

density, test duration, and extent of reduction in bacterial numbers is observed (Pankey and Sabath

2004).

There are over 22 classes of antibiotics and some examples of these classes include

penicillins, tetracyclines, cephalosporins, quinolones, lincomycins, macrolides, sulfonamides,

glycopeptides, aminoglycosides, and carbapenems with cephalosporins, penicillins, and quinolones

being the most successful (Coates et al. 2011). These classes are groupings of different antibiotics

that have similar chemical properties. Although there are many classes of antibiotics, the discovery

of new antibiotics has declined over the years (Coates et al. 2011).

Antibiotics produced by Bacillus

Many bacteria and fungi are known to produce small peptides, which attack bacteria and

have the potential to become an antibiotic compound, using non-ribosomal peptide synthesis, such

as Penecillium chrysogenum producing penicillin (Keszenman-Pereyra et al. 2003). The bacterial

genus Bacillus, is known to be able to produce more than 167 antibiotics (Stein 2005) (Table 1),

and, the species Bacillus subtilis is known to produce over two dozen antibiotics and lantibiotics

(Katz and Demain 1977). Lantibiotics are peptide antibiotics with inter-residual thioether bonds; in

other words, antibiotics that contain lanthionine, which occurs through post-translational

modification of ribosomally synthesized precursor peptides (Stein 2005).

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Bacillus strains contain antagonistic activity for pathogens such as bacteria and fungi

(Ongena and Jacques 2008). One type of antibiotic that Bacillus strains produce is a small peptide

that contains a long fatty acid chain called lipopeptide antibiotics, which are arranged in three

groups: the surfactin group, the fengycin group, and the iturin group (Ongena and Jacques 2008).

The difference between these groups is their chemical structure. Lipopeptides in the surfactin and

fengycin group contain a β -hydroxyl fatty acid; in the iturin group they contain a β-amino fatty acid

(Duitman et al. 1999). The iturin lipopetides also contain additional D-amino acids at position three

and six and also contain a tyrosine at the second amino acid position (Duitman et al. 1999).

Most of the antibiotics produced by Bacillus are synthesized by ribosomal and non-

ribosomal methods (Mannanov and Sattarova 2001). Compared to the traditional ribosomal

synthesis method, non-ribosomal synthesized peptides contain structural features such as D‐amino

acids, N‐ and C‐methylated amino acids, N‐terminally attached fatty acid chains, N formylated

residues, heterocyclic elements, glycosylated amino acids, and phosphorylated residues (Mannanov

and Sattarova 2001). Antimicrobial peptides (AMP) are natural antibiotics produced by bacteria

that induce chemokine production, accelerate angiogenesis, wound healing, and modulate

apoptosis in multicellular organisms that the bacteria infect (Guilhelmelli et al. 2013). AMPs have

been shown to increase cell membrane permeability, and many AMP’s have been shown to inhibit

protein, cell wall, and enzyme synthesis (Guilhelmelli et al. 2013).

Iturin A, mycosubtilin, flagellin, subtilin, translocation dependent antimicrobial spore

protein (TDASP), surfactin, and subtilosin A are antibiotics produced by Bacillus and are the

antibiotics of interest in this study. For this study, I focused on identifying ten genes that code for

enzymes known to be associated with antifungal and antibacterial pathways. These 10 genes were

Flagellin (hag), Mucosubtilin synthetase B (mycB), Translocation-dependent antimicrobial spore

component (tasA), 4’-phsophopantetheinyl transferase (surfactin) (sfp, sfpA, and sfpAA), Subtilin

(spaS1 and SpaS2), Iturin A (ituA), and Subtilosin A (sboA) (Yang et al. 2015, Velho et al. 2013,

Hassan et al. 2010, and Mora et al. 2011).

Iturin A is part of the iturin group and is a lipopeptide antibiotic that is synthesized non-

ribosomally by a large template of enzyme complexes (Tsuge et al. 2005). Genes involved in iturin

biosynthesis contain four open reading frames (ORFs) ituA, ituB, ituC, and ituD (Hassan et al. 2010)

(Figure 1). The ituD gene is responsible for β-amino acid synthesis, while the three larger genes

(ituA, ituB, and ituC) encode for enzymes for the synthesis of peptides (Tsuge et al. 2005). Iturin A is

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part of the lipopeptide antibiotic class (Duitman et al. 1999) and functions by binding to the

bacterial membrane directly causing cell death (Straus and Hancock 2006).

Mycosubtilin is also part of the iturin family and exhibits strong antifungal activity and

limited antibacterial activity (Duitman et al. 1999). Although the enzymes that are responsible for

the production of iturin lipopeptides are still unknown, it is known that it is similar to the antibiotic

surfactin and has an operon span of 38 kb of DNA (Duitman et al. 1999). However, it is known to be

the most active in the iturin family and has strong antifungal activity for yeast (Fickers et al. 2009).

Genes involved in mycosubtilin biosynthesis contain four open reading frames (ORFs) fenF, mycA,

mycB, and mycC (Duitman et al. 1999) (Figure 2). The fenF gene is responsible for β-amino acid

synthesis and the three larger genes (mycA, mycB, and mycC) encode for enzymes for the synthesis

of peptides (Tsuge et al. 2005). Antibiotics in the peptide class are a bacteriocins and inhibit cell

wall synthesis (McAuliffe et al. 2001).

Flagellin is a soluble mediator that causes inflammation in human intestines after an

infection occurs, which in turn induces cytokine release and impairs antigen presentation (Steiner

2007). Flagellin is a peptide antibiotic, meaning it functions by inhibiting cell wall synthesis

(McAuliffe et al. 2001). The flagellin gene, hag, with the help of 49 other genes, is involved in the

expression of flagella in prokaryotes (Totten and Lory 1990). Flagella provide motility to an

organism by having whip-like structures, and are recognized to be highly immunogenic (Steiner

2007). Flagellar structure is broken down into three architectural domains: the basal body, the

hook, and the filament (Mukherjee and Kearns 2014). Flagellin is composed of repeated protein

monomers that make up the filament. Flagellin is polymerized by the interaction between the N-

terminal and C-terminal domains (Mukherjee and Kearns 2014). There are four genes that are

involved in the production of the flagellin filament: fliD, hag, flgK, and flgL (Mukherjee and Kearns

2014) (Figure 3). The protein FliD catalyzes flagellin folding, serving as a filament cap, hag gene

encodes for flagellin monomer protein Hag and is essential for flagellar assembly, FlgK and FlgL

connect the hook and filament (Mukherjee and Kearns 2014).

Subtilin, a ribosomally synthesized peptide antibiotic (Klein and Entian 1994), is part of the

lanthionine antibiotic family and is involved in the post-translational modification of ribosomally

synthesized precursor peptides (Parisot et al. 2008). Subtilin is structurally related to Nisin, which

inhibits bacterial cell wall biosynthesis (Parisot et al. 2008). Subtillin is a lanthionine peptide

antibiotic, which protects against gram-positive bacteria and is also used in food preservation

(Klein and Entian 1994). Bacteriocins are ribosomally produced peptides produced by bacteria

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(Kawulka et al. 2004) and have bactericidal activity against Listeria monocytogenes (Thennarasu et

al. 2005). Genes involved in the subtilin biosynthesis include spaB, spaT, spaC, spaS, spaI, spaF, spaE,

spaG, spaR, and spaK (Stein 2005) (Figure 4). The spaB, spaT, spaC, and spaS are involved in the

post-transitional modification and transport, spaI, spaF, spaE, and spaG are involved in immunity,

spaR and spaK are the regulatory genes (Stein 2005). Antibiotics in the peptide class are

bacteriocins and inhibit cell wall synthesis (McAuliffe et al. 2001).

Translocation dependent antimicrobial spore protein (TDASP) gene, tasA, is involved in the

production of the 31-kDA protein Tas A (Stover and Driks 1999). TasA has antibacterial activity

against gram-positive and gram-negative bacteria (Stover and Driks 1999). It also has a broad

spectrum antibacterial activity meaning it can target a bigger range of bacterial species (Stover and

Driks 1999). Genes involved in the TDASP biosynthesis include 3 open reading frames (ORFs) tasA,

yqxM, and sipW (Stover and Driks 1999) (Figure 5). sipW is involved in secretion, tasA is involved in

germination (Stover and Driks 1999) and yqxM is involved in growth (Serrano et al 1999). TasA is

known to be secreted by spores allowing for inhibition of possible competing bacteria during and

after sporulation (Stover and Driks 1999). TDASP is a peptide antibiotic (Stover and Driks 1999)

which means that it functions by inhibiting cell wall synthesis (McAuliffe et al. 2001).

The 4’-phosphopantetheinyl transferase, gene sfp, which produces surfactin, is a detergent-

like lipopeptide that reduces surface tension of water and possesses hemolytic, antiviral,

antibacterial, and antitumor properties (Heerklotz and Seelig 2001). Surfactin is a lipoheptapeptide

antibiotic (Quadri et al. 1998) and consists of heptapeptides that contain a β-hydroxy fatty acid

with 13 to 15 carbons atoms (Hassan et al. 2010). Genes involved in the surfactin biosynthesis

contain four open reading frames (ORFs) srfA-A, srfA-B, srfA-C, srfA-D (Hassan et al 2010) (Figure 6).

The srfAA gene encodes enzyme E1A, srfAB encodes enzyme E1B, srfAC encodes enzyme E2, and

srfAD encodes thioesterase-like protein and sfp, which is required for the activation by

posttranslational for surfactin synthetase (Vollenbroich et al. 1994 and Peypuc et al. 1999).

Surfactin excretion is still not fully known and an active transporter has not been found, therefore,

implying passive diffusion across the cytoplasmic membrane (Heerklotz and Seelig 2001). The

antibiotic class lipopeptide work by disrupting cell membrane and eventually causes cell death

(Straus and Hancock 2006).

Subtilosin A is the only anionic, circular antimicrobial peptide and is also a bacteriocin

(Velho et al. 2013, Huang et al. 2009). More specifically gene, sboA, encodes for a 43-amino-acid

subtilosin A precursor and is found downstream of genes albABCDEF: ablA, albB, albC, albD, albE,

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and albF (Huang et al. 2009) and sboX, which a newly discovered gene, the function of which is still

unclear (Zheng et al. 2000) (Figure 7). ablA, albE, albF, and sboA are involved in the post-

transitional modification and transport, albB, albC, and albD are involved in immunity (Stein 2005).

Subtilosin A is in the antibiotic class peptides (Stein 2005), which are bacteriocins that work by

targeting cell wall synthesis, DNA replication and transcription, and disrupting the bacterial

membrane (Cavera 2014).

Previous work (Szick et al. 201X) isolated bacteria from the skin of the North American

bullfrog and the California toad. The samples were challenged against five environmental fungi

using challenge assays in order to identify bacteria with these genes that have been known to

contain antifungal properties. The results showed that 64 of the 225 bacteria collected inhibited the

growth of at least one environmental fungus (Szick et al. 201X). Of the 64 isolates, 17 were

identified as Bacillus. Using DNA, those bacterial isolates were identified at the genus level using

PCR and DNA sequencing. In order to investigate the presence of the genes known to be involved in

the antibiotic producing pathways in Bacillus bacteria Zepeda (2016) tested the presence of bamC,

fend, ituD, srfDB3, ofr2, and zmaR. These genes code for enzymes involved in the production of

bacillomycin D, fengycin, iturin A, surfactin, and zwittermicin A. Zepeda (2016) obtained PCR

product in isolate 664 for orf2 and zmaR, which are involved in the production of Zwittermicin A

and negative results for the remainder of the antibiotics.

In order to further investigate the presence of these antibiotic biosynthetic pathways, this

study focused on ten genes and the identification of genes that code for enzymes in these antifungal

and antibacterial pathways; Flagellin (hag), Mucosubtilin synthetase B (mycB), Translocation-

dependent antimicrobial spore component (tasA), 4’-phsophopantetheinyl transferase (surfactin)

(sfp, sfpA, and sfpAA), Subtilin (spaS1 and Spas2), Iturin A (ituA), and Subtilosin A (sboA) (Yang et al.

2015, Velho et al. 2013, Hassan et al. 2010, and Mora et al. 2011). I hypothesize that our 16 Bacillus

samples will contain genes that are known to be involved in known antibiotic biosynthetic

pathways.

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Table 1. A list of some of the antibiotics produced by Bacillus species (Modified from Stein 2005).

Species Antibiotics

Bacillus brevis Linear gramicidin

Bacillus subtillis Subtilin

Mycosubtilin

Iturin

Bacillus licheniformis Bacitracin

Bacillus polymyxa Polymyxin

Bacillus circulans Xylostatin

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itu 38.0 kb

D A B C

Figure 1. The organization of gene clusters of all the genes involved in the production of iturin A (ituA, ituB, ituC, and ituD)

with base pair length (kilobase (kb)) (Modified from Stein 2005). The ituD gene is responsible for β-amino acid synthesis and

the three larger genes (ituA, ituB, and ituC) encode for enzymes for the synthesis of peptides (Modified Tsuge et al. 2005).

myc 38.0 kb

fenF A B C

Figure 2. The organization of gene clusters of all the genes involved in the production of mycosubtilin (fenF, mycA, mycB, and

mycC) with base pair length (kilobase (kb)) (Modified from Stein 2005). The fenF gene is responsible for β-amino acid

synthesis and the three larger genes (mycA, mycB, and mycC) encode for enzymes for the synthesis of peptides (Modified from

Tsuge et al. 2005).

B C

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hag 4.4 kb

flgK flgL hag fliD

Figure 3. All the genes involved in the production of Flagellin (fliD, hag, flgK, and flgL) (Modified MukherJee and Kearns 2014)

with an approximate base pair length (kilobase (kb)) (Modified from Soldo et al. 1996). The protein FliD catalyzes flagellin

folding, serving as a filament cap, hag gene encodes for flagellin monomer protein Hag and is essential for flagellar assembly,

FlgK and FlgL connect the hook and filament (Modified from Mukherjee and Kearns 2014).

spa 12.0 kb

B T C S I F E G R K

Figure 4. Bacillus subtilis lantibitoics involved in the production of subtilin (spaB, spaT, SpaC, spaS, spaI, spaF, spaE, spaG, spaR,

and spaK) Color code: Black: genes involved in post-transitional modification and transport, marble: immunity genes, and

gray: regulatory genes. Number of kilobases (kb) is the size of the gene cluster (Modified from Stein 2005)

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tasA 1.3 kb

yqxM sipW tasA

Figure 5. The organization of gene clusters of all the genes involved in the production of TDA (yqxM, sipW, and tasA) (Modified

Stover and Driks 1999) with an approximate base pair length (kilobase (kb)) (Modified from Chu et al. 2002). The sipW is

involved in secretion, tasA is involved in germination (Stover and Driks 1999), yqxM is involved in growth (Modified from

Serrano et al. 1999).

srf 31.0 kb srfAA srfAB srfAC srfA-D sfp

Figure 6. The organization of gene clusters of all the genes involved in the production of the antibiotic surfactin (srfA-A, srfA-B, srfA-C, srfA-D, and sfp) with base pair length (kilobase (kb)) (Modified from Stein 2005 and modified from Das et al. 2008). The srfA-A gene encodes enzyme E1A, srfA-B encodes for enzyme E1B, srfA-C encode for enzyme E2, and srfA-D encodes for thiosterase-like protein (Vollenbroich et al. 1994) and sfp which is required for the activation by posttranslational for surfactin synthetase (Modified Das et al. 2008 and Peypuc et al. 1999).

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alb 7.0 kb

sbo A B C D E F G

Figure 7. Bacillus subtilis lantibitoics involved in the production of subtilosin A (sbo, ablA, albB, albC, albD, albE, albF, and

albG). Color code: Black: genes involved in post-transitional modification and transport, marble: immunity genes, and white:

regulatory genes. Number of kilobases (kb) is the size of the gene cluster (Modified from Stein 2005).

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Methods

Cultivation of Bacterial Isolates

Bacterial isolates were previously collected in the Szick and Lauer labs’ from 4 North American

bullfrogs (Lithobates catesbeiana) and 5 California toads (Anaxyrus boreas halophilus) from the

Bakersfield, CA area. Bacterial isolates were grown using glycerol stocks from the bacterial culture

collection in the Szick laboratory (Szick et al 201X). The bacterial isolates were streaked on R2A plates

and incubated at 37°C for 48 hours. A single colony from each plate was re-streaked on a new plate to

ensure pure cultures. Positive control strains (10A23, 10A6, 2A9, and 1A747) used in PCR analysis were

obtained from the Bacillus Genetic Stock Center at Ohio State University in the form of a dried filter disk

(Table 2). Each disk was placed on blood agar and TSB agar and two drops of TSA broth was dropped on

each disk. The plates were incubated at 37°C for 48 hours. A single colony from each plate was re-

streaked on a new R2A plate in order to ensure pure cultures.

DNA Extractions and PCR

DNA extractions were performed from plates that contained pure colonies with Ultraclean DNA

isolation kit (MoBio Laboratories) following the manufacturer’s protocol. PCR primers were purchased

from INTEGRATED DNA TECHNOLOGIES (IDT). PCR was performed on all samples with positive controls

using 8F/1492R in order to amplify the 16S rRNA gene. To test the presence or absence of the target

genes a set of ten primers were synthesized and used in subsequent PCR reactions (Table 2).

PCR was carried out in a total volume of 28 μl containing 11.5 μl GoTaq polymerase (Promega

Corporation), 0.15 nmoles of each forward and reverse primer, 10.5 μl of water, and 5μl of DNA.

Thermocycler conditions for all primers are shown in Table 3, except mycB, presented by Yang et al.

2015, the annealing temperatures and number of cycles were modified from the original reference.

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Seven microliters of each PCR sample was loaded into a 1% agarose gel that contained Syber safe stain

for electrophoresis analysis for 20-30 minutes at 150 volts and imaged (Universal Hood 2, Biorad).

PCR Product Purification and Sequencing

According to the manufacturers protocol, positive samples and positive controls were purified

using ExoSAP-IT PCR clean-up kit (Affymetrix). Purified samples were sequenced at Laragen Sequencing

and Genotyping in Culver City, California (http://laragen.com/). Sequences were verified using NCBI

nBlast search for nucleotides (https://blast.ncbi.nlm.nih.gov/Blast.cgi).

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Table 2. Specific primer sets for the detection of antibiotic biosynthetic genes involved in the production of the antibiotics flagellin,

mycosubtilin, translocation-dependent antimicrobial spore component (TDA), surfactin, subtilin, iturin A, and subtilosin A.

Antibiotic Gene Reference Strain Sequence (5’-3’) Primer Name Product Reference name length (bp)

Flagellin hag B. subtilis 10A23 ATGAGAATCAACCACAATATCGC hagF 1,210 Yang et al. 2015 TTAACCTTTAAGCAATTGAAGAAC hagR

Mycosubtilin mycB B. subtilis 6633 ATGTCGGTGTTTAAAAATCAAGTAACG mycBF 2,024 Yang et al. 2015 TTAGGACGCCAGCAGTTCTTCTATTGA mycBR

TDA tasA B. subtilis 10A23 ATGGGTATGAAAAAGAAATTAAG tasAF 786 Yang et al. 2015 TTAGTTTTTATCCTCACTGTGA tasAR

Iturin A ituA B. subtilis 10A6 ATGAAAATTTACGGAGTATATATG ituAF 1,150 Yang et al. 2015 TTATAACAGCTCTTCATACGTT ituAR

Subtilosin A sboA B. subtilis 6633 CATCCTCGATCACAGACTTCACATG sboAF 734 Velho et al. 2013 CGCGCAAGTAGTCGATTTCTAACAC sboAR

Subtilin spaS1 B. subtilis 2A9 ATGTCAAAGTTCGATGATTTCGA spaS1F 290 Yang et al. 2015 TTATTTAGAGATTTTGCAGTTACA spaS1R

spaS2 B. subtilis 2A9 TGTCATGGTTACAGCGGTATCGGTC Spas2F 566 Velho et al. 2013 AGTGCAAGGAGTCAGAGCAAGGTGA Spas2R

Surfactin srfAC B. subtilis 10A6 GATCAGGTTCARGAYATGTATTA srfACF 3,700 Hassan et al. 2010 AGCATTTCTGCGTGYGTKCC srfACR

srfAA B. subtilis 10A6 TCGGGACAGGAAGACATCAT SRFAF 201 Mora et al. 2011 CCACTCAAACGGATAATCCTGA SRFAR

sfp B. subtilis 10A6 ATGAAGATTTACGGAATTTATATG sfpF 675 Yang et al. 2015 TTATAAAAGCTCTTCGTACGAG sfpR

NA 16S AGAGTTTGATCCTGGCTCAG 8F 1,600 Eden et al. 1991 rRNA GGTTACCTTGTTACGACTT 1492R

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Table 3. Thermocycler conditions for each primer pair.

Primer Pair Initial Denaturation Annealing Extension # of Final Source Denaturation cycles Extension

hagF/R 95°C, 3m 95°C, 30s 50°C, 1m 72°C, 90s 35 72°C, 5m Yang et al. 2015

mycBF/R 95°C, 3m 95°C, 30s 55°C, 1m 72°C, 90s 30 72°C, 5m Yang et al. 2015

tasAF/R 95°C, 3m 95°C, 30s 45°C, 1m 72°C, 90s 35 72°C, 5m Yang et al. 2015

sfpF/R 95°C, 3m 95°C, 30s 49°C, 1m 72°C, 90s 35 72°C, 5m Yang et al. 2015

spaS1F/R 95°C, 3m 95°C, 30s 49°C, 1m 72°C, 90s 35 72°C, 5m Yang et al. 2015

ituAF/R 95°C, 3m 95°C, 30s 49°C, 1m 72°C, 90s 35 72°C, 5m Yang et al. 2015

sboAF/R 95°C, 3m 95°C, 30s 50°C, 1m 72°C, 90s 35 72°C, 5m Velho et al. 2013

spaS2F/R 95°C, 3m 95°C, 30s 55°C, 1m 72°C, 90s 35 72°C, 5m Velho et al. 2013

srfACF/R 95°C, 5m 94°C, 1m 55°C, 3m 72°C, 3m 30 72°C, 5m Hassan et al 2010

srfAAF/R 95°C, 4m 94°C, 1s 58°C, 1m 70°C, 1m 40 70°C, 5m Mora et al 2011

8F/1492R 95°C, 3m 95°C, 1m 60°C, 1m 72°C, 2m 35 72°C, 3m Brunel et al 1997

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Results

Confirmation of bacterial isolate species

All 16 isolates were confirmed as bacteria by using primer pair 8F/1492R to amplify the 16S

rRNA gene. A positive amplicon signified the DNA extractions worked and that all 16 isolates were

bacteria (Figures 8, 9, and 10).

Confirmation of positive control and PCR analysis

All primer pairs had a positive control by obtaining a Bacillus strain that possessed genes that

code for enzymes involved in the production of flagellin, mycosubtilin, translocation-dependent

antimicrobial spore component (TDA), surfactin, subtilin, iturin, and subtilosin PCR was performed with

primer pairs specific for genes in the pathways listed above. Table 4 shows a summary of these results.

All 16 isolates were negative for detection of genes tasA, mycB, ituA, hag, and spaS2 (Figures 11,

13, 15, 17, and 34). The positive controls for the genes were successfully amplified, sequenced, and

confirmed with nBlast (Figure 12, 14, 16, and 18).

The specific primer pair, sboAF/R, was used to screen 16 Bacillus isolates for the gene that is

involved in the biosynthesis of subtilosin A. Isolates 551,615,616, 620, and 664 were positive for the

sboA gene (Figure 19). The positive control for sboA was successfully amplified, sequenced, and

confirmed with nBlast (Figure20). Isolate 551 was sequenced and nBlast confirmed the presence of the

sboA gene (Figure 21).

The specific primer pair, sfpF/R, was used to screen 16 Bacillus isolates for the gene that is

involved in the biosynthesis of surfactin. Isolates 517, 551, and 620 were positive for the sfp gene

(Figure 22). Isolate 551 was sequenced and nBlast confirmed the presence of the sfp gene (Figure 23).

The positive control for sfp was successfully amplified, sequenced, and confirmed with nBlast (Figure

24).

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To further investigate the presence of other genes, that are also involved in the production of

surfactin in the specific primer pair, srfACF/R and srfAAF/R, were used to screen 16 Bacillus isolates for

the gene that is involved in the biosynthesis of Surfactin. Isolates 615 and 620 were positive for the

sfpAC gene (Figure 25). The positive control for srfAC was successfully amplified, sequenced, and

confirmed with nBlast (Figure 26). Isolate 620 was sequenced and nBlast confirmed the presence of the

sfpAC gene (Figure 27). Isolates 517, 539, 551, 620, 664, and 730 were positive for the sfpA gene (Figure

28). The positive control for sfpA was successfully amplified, sequenced, and confirmed with nBlast

(Figure 17). Isolate 551 was sequenced and nBlast confirmed the presence of the sfpA gene (Figure 30).

The specific primer pair, SpaS1F/R, was used to screen 16 Bacillus isolates for the gene that is

involved in the biosynthesis of Subtilin. Isolate 616 was positive for the spaS1 gene (Figure 31). The

positive control for SpaS1 was successfully amplified, sequenced, and confirmed with nBlast (Figure 32).

Isolate 616 was sequenced and nBlast confirmed the presence of the spaS gene (Figure 33).

Our 16 Bacillus isolates were negative for four of our antibiotics (Flagellin, mycosubtilin, TDA,

and, iturin A). Also, eight of our isolates (524, 544, 585, 586, 623, 626, 672, 727) were negative for all of

our antibiotics. Isolate 620 had the most positive results out of our 16 isolates showing that it contains

genes that code for enzymes involved in the production of subtilosin A and surfactin.

22

Table 4. A summary of the results for specific primer sets for the detection of antibiotic biosynthetic genes involved in the production of the

antibiotics flagellin, mycosubtilin, translocation-dependent antimicrobial spore component (TDA), surfactin, subtilin, iturin A, and

subtilosin A. Those with a X had a positive.

Antibiotic

Flagellin

Isolate Gene

hag

517 524 539 544 551 585 586 615 616 620 623 626 664 672 727 730

Mycosubtilin mycB

TDA tasA

Iturin A ituA

Subtilosin A sboA X X X X X

Subtilin spas

spaS X

Surfactin sfp X X X

srfAC X X

srfA X X X X X X

23

bp

517 524 539 544 585 586 616 623 626 664 672 727 NC

1484 bp

Figure 8. Agarose gel electrophoresis screening for the presence of Bacillus Spp. Amplified with 8F/1492R (8F: 5’-AGA GTT TGA TCC TGG CTC AG-3’ and 1492R: 5’- CGG TTA CCT TGT TAC GAC TT-3’) in samples 1-12 of the 16 bacterial isolates, negative control (N), and HINDIII ladder visualized using a 1% agarose gel with 0.01% SYBR Safe. The expected fragment length is 1484 bp.

24

bp 524 539 551 585 615 616

1484 bp

1484 bp

Figure 9. Agarose gel electrophoresis screening for the presence of Bacillus Spp. Amplified with 8F/1492R (8F: 5’-AGA GTT TGA TCC TGG CTC AG-3’ and 1492R: 5’- CGG TTA CCT TGT TAC GAC TT-3’) in samples 1-12 of the 16 bacterial isolates, negative control (NC), and HINDIII ladder visualized using a 1% agarose gel with 0.01% SYBR Safe. The expected fragment length is 1484 bp.

517 730 NC

25

bp

620 620 NC

1484 bp

Figure 10. Agarose gel electrophoresis screening for the presence of Bacillus Spp. Amplified with 8F/1492R (8F: 5’-AGA GTT TGA TCC TGG CTC AG-3’ and 1492R: 5’- CGG TTA CCT TGT TAC GAC TT-3’) in samples 1-12 of the 16 bacterial isolates 620 on new R2A agar (1), 620 on old R2A agar (2), negative control (NC), and HINDIII ladder visualized using a 1% agarose gel with 0.01% SYBR Safe. The expected fragment length is 1484 bp.

26

bp

620 623 626 664 672 727 730 NC PC

786 bp

517 524 539 544 551 585 586 615 616 Figure 11. Agarose gel electrophoresis screening for the presence of Bacillus Spp. Amplified with TasA (F:

5’- ATGGGTATGAAAAAGAAATTAAG -3’ and R: 5’- TTAGTTTTTATCCTCACTGTGA -3’) in samples of the 16 bacterial isolates that were used, negative control (NC), positive control (PC), and lambda DNA/HINDIII

ladder visualized using a 1% agarose gel with 0.01% SYBR Safe. The expected fragment length is 786 bp.

27

Figure 12. NCBI nBlast sequence alignment of the positive control for gene TasA. Identification was 95%

to Bacillus amyloliquefaciens CC178, complete genome with a subject number of 2424246 and sequence

ID (CP006845.1) states that that subject number produces TasA.

28

620 623 626 664 672 727 730 NC PC bp

2024 bp

517 524 539 544 551 585 586 615 616

Figure 13. Agarose gel electrophoresis screening for the presence of Bacillus Spp. Amplified with mycB

(F: 5’- ATGTCGGTGTTTAAAAATCAAGTAACG -3’ and R: 5’- TTAGGACGCCAGCAGTTCTTCTATTGA -3’) in samples of the 16 bacterial isolates that were used, negative control (NC), positive control (PC), and

lambda DNA/HINDIII ladder visualized using a 1% agarose gel with 0.01% SYBR Safe. The expected

fragment length is 2,024 bp.

Quer y 3 TGT- ATCCCGTATTTTAAA- C- CCGG- AAACGCATCATTAACCCGTGTTGGCTATCAAGA 58 I l l 111111111111111 I I l l 111111111111111111111111111111 I I

Sbj c t 1 935893 TGTAATCCCGTATTTTAAAACAGCGGAAAACGCATCATTAACCCGTGTTGGCTATCAGGA 1 935834

Quer y 59 AAAAAGCATCTATCGCTCCTTATCTCCAGAAGTATCTCAAAGAATATTGACAATGGCTAA 1 18 111111111111111111111111111111111111111111111111111111111111

Sbj c t 1 935833 AAAAAGCATCTATCGCTCCTTATCTCCAGAAGTATCTCAAAGAATATTGACAATGGCTAA 1 935774

Quer y 1 1 9 TCATTCCGAAATGG~ACCTATTTGATTTTATTGGCAGGCATC~ATGTTTGTTGTATAA 178 111111111111111111111111111111111111111111111111111111111111

Sbj c t 1 93577 3 TCATTCCGAAATGG~ACCTATTTGATTTTATTGGCAGGCATC~ATGTTTGTTGTATAA 1 935714

Quer y 1 7 9 ATATACAGATCGAGCGAGCACGATTCTGGGTATTC~ACGGTATCTAAGCAAGTT-GCAG 237 1111111111111111111111111111111111111111111111111111 1111

Sbj c t 1 93571 3 ATATACAGATCGAGCGAGCACGATTCTGGGTATTC~ACGGTATCTAAGCA~~a~~GCAG 1 935654

Quer y 238 CTCTTCAACTGTCAATACCATTGTCCTATTGAAGAATACCCTAATCTGCCAAAGTACATT 297 111111111111111111111111111111111111111 I I I 111111111111111

Sbj c t 1 935653 CTCTTCAACTGTCAATACCATTGTCCTATTGAAGAATACGCTTAGCTGCCAAAGTACATT 1 935594

Quer y 298 TAAAACCGTGTTCGAACAACTaaaaaaaGCGGTTAATGATTCGCTA~~aaaTCCGAACCT 357 11111111111111111111111111111111111111111111111111111 I I I I I

Sbj c t 1 935593 TAAAACCGTGTTCGAACAACTA~aaa~~GCGGTTAATGATTCGCTA~~aaaT~CCT 1 935534

Quer y 358 GCCTTTTCG~AATCGGACAACATGTAGATGTGCAATATTACCACCAGAACATCCCGGT 417 11111111111111111 I I 111111 1111111111 I I I I 11111111111 I

Sbj c t 1 935533 GCCTTTTCGAAAAATCGTTCAGCATGTAAATGTGCAATACGACAACGAGAACATCCCGTT 1 93547 4

Quer y 418 AATTCATAGCGTTGTTTCGCTCATTGAAATTCATTCCTTGCAATTTAAGGAAGACNTTGN 477 11111111 11111111111111 1111111111111111111111111111111 I l l

Sbj c t 1 93547 3 AATTCATACCGTTGTTTCGCTCAATGAAATTCATTCCTTGCAATTTAAGGAAGACATTGC 1 935414

Quer y 478 ANCTGATACGNTGTTTCATTTTGACTTGGAGAATANCNAAATTCATTTGAAACTTATTTA 537 I 11111111 111111111111111111111111 I 1111111111111111111111

Sbj ct 1 93541 3 AACTGATACGTTGTTTCATTTTGACTTGGAGAATAGCGAAATTCATTTGAAACTTATTTA 1 935354

Quer y 5 38 TAACGG~ATCTTTATGATGAGGACTATATGGACC~ATGGTGTCCCATCTCAATCANCT 597 111111111111111111111111111111111111111111111111111111111 I I

Sbj ct 1 935353 TAACGG~ATCTTTATGATGAGGACTATATGGACC~ATGGTGTCCCATCTCAATCAGCT 1 935294

Quer y 598 GCTGTCCGTGATCTTGTTCCANCCCCAAGCTGCAATCCNTACAGCANAANGCATACCCTC 657 111111111111111111111 1111111111111111 1111111 I I 11111111

Sbj ct 1 935293 GCTGTCCGTGATCTTGTTCCAGCCCCAAGCTGCAATCCATACAGCAGAAGGCATACCCG- 1 935235

Quer y 658 AANCGGTCAAACAN~AATTTTGTTTGACTTT~~TGACACCGCCNCANATTANTCTGGAA 717 I I 1111111111 1111111111111111111 1111111111 I I 1111 1111111

Sbj ct 1 935234 AAGCGGTCAAACAG~AATTTTGTTTGACTTTAATGACACCGCCGCAGATTATTCTGGAA 1 9351 7 5

Quer y 718 NCNCNACATTCAGTCGATTATTTGAAGANCANGCGG~AAGAACNCANGATCATGTANCTG 7 77 I I l l I 1111 111111111111 I I 11111111111 I 111111111 I l l

Sbj ct 1 9351 7 4 ACAAAACAGTAAGT~ATTATTTGAAGAGCAGGCGG~AA~ACGCCTGATCATGTAGCTG 1 935115

Quer y 778 TNAANTTCGTTAACANTCANATGACATACANAGAANTGAATGAT~AATCTAATCNTTTGG 837 I I I 1111111111 I l l 1111111111 1111 1111111 1111111111 I I I I I

Sbjct 193511 1 TTAACTTCCTTAACAATCATATCACATACACACAATTCAATCA~AATCTAATCCTTTCc 1 935055

Quer y 838 CCAGAACGTTGCAAAACTCCG- TGTTCNNGCCGATACNTTGATCNCCATCATNNNNGANC 896 I 1111111111 I I I I I I I I I I I I 11111111 I l l I I 1111111 I I I

Sbj ct 1 935054 CAAGAACGTTGCGAAACTACGGTGTTCAAGCCGATACATTGGTCGCCATCATGGCAGAGC 1 934995

Quer y 897 GTTCNTT~~ANN-GATCGTGTCCATC 921 1111 I l l I 1111111111111

Sbj ct 1 934994 GTTCGTTAGAAATGATCGTGTCCATC 1 934969

29

Figure 14. NCBI nBlast sequence alignment of the positive control for gene mycB. Identification was 91%

to Bacillus subtilis subsp. Spizizenil str W23, complete genome with a subject number 1935893 and

sequence ID (CP002813.1) states that that subject number produces Mycosubtilin synthase subunit B.

30

bp

620 623 626 664 672 727 730 NC PC

1150 bp

517 524 539 544 551 585 586 615 616

Figure 15. Agarose gel electrophoresis screening for the presence of Bacillus Spp. Amplified with ituA (F:

5’- ATGAAAATTTACGGAGTATATATG -3’ and R: 5’- TTATAACAGCTCTTCATACGTT -3’) in samples of the 16 bacterial isolates that were used, plus negative control (NC), positive control (PC), and lambda

DNA/HINDIII ladder visualized using a 1% agarose gel with 0.01% SYBR Safe. The expected fragment

length is 1,150 bp.

31

Figure 16. NCBI nBlast sequence alignment of the positive control for gene ituA. Identification was 99%

to Bacillus subtilis strain RP24 lipopetide antibiotic iturin A gene, complete genome with a subject

number 18 and sequence ID (EU797520.1) states that that subject number produces lipopetide

antibiotic iturin A.

32

bp 620 623 626 664 672 727 730 PC NC

1,210 bp

517 524 539 544 551 585 586 615 616

Figure 17. Agarose gel electrophoresis screening for the presence of Bacillus Spp. Amplified with hag (F:

5’- ATGAGAATCAACCACAATATCGC -3’ and R: 5’- TTAACCTTTAAGCAATTGAAGAAC -3’) in samples of the 16 bacterial isolates that were used, negative control (NC), positive control (PC), and lambda

DNA/HINDIII ladder visualized using a 1% agarose gel with 0.01% SYBR Safe. The expected fragment

length is 1,210 bp.

Query 7 AGCCTGAANAAGGATGTTGTTTTTCGTGTGCTCCATCATCTCAGAAGCCATGTCTACGTC 66 1111111 11111111111111111111 111111111111111111111111111111

Sbjct 3021978 AGCCTGAGTAAGGATGTTGTTTTTCGTGTACTCCATCATCTCA~AGCCATGTCTACGTC 3 02191 9

Query 6 7 ACGGATACGTGATTCAGCAGAAGTCAGGTTCTCAGAAGAAGTACCAAGGrTGTTGATTGT 126 111111111111111111111111111111111111111111111111111111111111

Sbjct 3021918 ACGGATACGTGATTCAGCAGAAGTCAGGTTCTCAGAAGAAGTACCAAGGrTGTTGATTGT 3 021859

Query 127 GTGCTCT~AACGGTTTTGAACTGCACCAAGTTTAGCGCGCTCGCTAGATACTGTGTCGAT 1 86 111111111111111111111111111111111111111111111111111111111111

Sbjct 3021858 GTGCTCTAAACGGTTTTGAACTGCACCAAGTTTAGCGCGCTCGCTAGATACTGTGTCGAT 3 021799

Que~y 1 07 AGCTGTTTTGATTGTTGTAAGAGCTGAAGACGCAGCTTTAGCTGAAGAT3AGATGTCGAT 24G 111111111111111111111111111111111111111111111111111111111111

Sbjct 3021798 AGCTGTTTTGATTGTTGTAAGAGCTGAAGACGCAGCTTTAGCT~AGAT3AGATGTCGAT 3 021739

Query 247 ACCTTTTGTTACTTTAGAATCTGCAGCTAATTTTTCAGATGCAACAACTr TACCTTTATC 3 06 111111111111111111111111111111111111111111111111111111111111

Sbjct 3021738 ACCTTTTGTTACTTTAGAATCTGCAGCTAATTTTTCAGATGCAACAACTr TACCTTTATC 3 0216 79

Query 307 ATCATAATATCCAGCAGCTTTTGTGACTTCACCAGTAGTTCCGTCTTTTrCTTCGTCAGC 366 111111111111111111111111111111111111111111111111111111111111

Sbjct 30216 78 ATCATAATATCCAGCAGCTTTTGTGACTTCACCAGTAGTTCCGTCTTTTrCTTCGTCAGC 3 02161 9

Query 367 CCAAGTAGCAGTGCTTCCATCAGTTGCTGTAAGTGTATTTTGATCACCA3ATACAGTATA 426 111111111111111111111111111111111111111111111111111111111111

Sbjct 3021618 CCAAGTAGCAGTGCTTCCATCAGTTGCTGTAAGTGTATTTTGATCACCA3ATACAGTATA 3 021559

Query 427 AGTCGTACCAACTTTCAGGCTTTCAAAGTCCATTTTATTGATAGACAGG:TCATTGTTTG 486 1111111111111111111111111 1111111111111111111111111111111111

Sbjct 3021558 AGTCGTACCAACTTTCAGGCTTTCAGAGTCCATTTTATTGATAGACAGG:TCATTGTTTG 3 021499

Query 487 GCCTTCGTTAGCTCCGATTTGGAACGTAAGGTTTTGCGCAGTTCCGTCA~GAAGTTTCTT 546 111111111111111111111111111111111111111111111111111111111111

Sbjct 3021498 GCCTTCGTTAGCTCCGATTTGGAACGTAAGGTTTTGCGCAGTTCCGTCA~GAAGTTTCTT 3 021439

Query 547 CGTATTGAACTCAGTGTCAGTAGAGATTCTTGTTACTTCAGACGCTAATrGGTCCATCTC 6 06 111111111111111111111111111111111111111111111111111111111111

Objet 3 0 2143 0 CGTATTG~ACTCAGTGTCAGTAGAGATTCTTGTTACTTCAGACGCTAATrGGTCCATCTC 3 0 213 7 9

Query 6 07 TTTTTGAAGCTCAGAACGGTCANAATCAGTGTTTGTATCGTTCGCCGCTrGTGTA~AG 666 1111111111111111111111 1111111111111111111111111111111111111

Sbjct 3021378 TTTTTGAAGCTCAGAACGGTCAGAATCAGTGTTTGTATCGTTCGCCGCTrGTGTA~AG 3 02131 9

Query 667 CTCGCTCATACGCTGAAGAATGCTGTGAGTTTCGTTCAATGCACCCTCA3ATGTTTGGAT 726 111111111111111111111111111111111111111111111111111111111111

Sbjct 3021318 CTCGCTCATACGCT~AGAATGCTGTGAGTTTCGTTCAATGCACCCTCA3ATGTTTGGAT 3 021259

Query 727 AAGAGAGATTCCGTCTTGAGCATTTTTAGAAGCCATGTCT~AACCGCGGATTTGGGAACG 786 111111111111111111111111111111111111111111111111111111111111

Sbjct 3021258 AAGAGAGATTCCGTCTTGAGCATTTTTAGAAGCCATGTCT~AACCGCGGATTTGGGAACG 3 0211 99

Query 787 CATTTTTTCAGAGATCGCAAGANCCGCAGCGTCATCACCAGCGCGGTTGATGCGAAGACC 846 1111111111111111111111 1111111111111111111111111111111111111

Sbjct 30211 98 CATTTTTTCAGAGATCGCAAGACCCGCAGCGTCATCACCAGCGCGGTTGATGCGAAGACC 3 0211 39

Query 847 TGAAGAT~ATTTTTCCATGTTTTTTGCAGCAGAGTTTGAACCTGCATTCANCTGACGGCT 906 11111111111111111111111111111111111111111111111111 111111111

Sbjct 30211 38 TGAAGAT~ATTTTTCCATGTTTTTTGCAGCAGAGTTTGAACCTGCATTCAGCTGACGGCT 3 021079

Query 907 AGTGTTAAGAGCCGCNATATTGNGNTGAATTCTCA 94 1 111111111111111 111111 I I 1111111

Sbjct 3021078 AGTGTTAAGAGCCGCGATATTGTGGTTGATTCTCA 302104 4

33

Figure 18. NCBI nBlast sequence alignment of the positive control for gene hag. Identification was 99%

to Bacillus amyloiquefaciens strain WS-8, complete genome genome with a subject number 3021978

and sequence ID (CP018200.1) states that that subject number produces Flagellin.

34

bp 620 623 626 664 672 727 730 PC NC

734 bp

734 bp

517 524 539 544 551 585 586 615 616 Figure 19. Agarose gel electrophoresis screening for the presence of Bacillus Spp. Amplified with sboA

(F: 5’- CATCCTCGATCACAGACTTCACATG -3’ and R: 5’- CGCGCAAGTAGTCGATTTCTAACAC -3’) in samples

of the 16 bacterial isolates that were used, negative control (NC), positive control (PC), and lambda

DNA/HINDIII ladder visualized using a 1% agarose gel with 0.01% SYBR Safe. The expected fragment

length is 734 bp.

35

Figure 20. NCBI nBlast sequence alignment of the positive control for gene sboA. Identification was 96%

Bacillus subtilis subsp. Spizizenii strain DSM 15029 subtilosin A gene clucter, partial sequence with a

subject number 129 and sequence ID (JN118835.1) states that that subject number produces sboA.

36

Figure 21. NCBI nBlast sequence alignment of isolate 551 with primer sboA. Identification was 99% to

Bacillus sp. LM 4-2, complete genome with a subject number 3689194 and sequence ID (CP011101.1)

states that that subject number produces subtilin.

37

bp

620 623 626 664 672 727 730 NC PC

675 bp

675 bp

517 524 539 544 551 585 586 615 616 Figure 22. Agarose gel electrophoresis screening for the presence of Bacillus Spp. Amplified with sfp (F:

5’- ATGAAGATTTACGGAATTTATATG -3’ and R: 5’- TTATAAAAGCTCTTCGTACGAG -3’) in samples of the 16 bacterial isolates that were used, negative control (NC), positive control (PC), and lambda DNA/HINDIII

ladder visualized using a 1% agarose gel with 0.01% SYBR Safe. The expected fragment length is 675 bp

38

Figure 23. NCBI nBlast sequence alignment of the positive control for gene sfp. Identification was 99% to

Bacillus subtilis strain T30, complete genome genome with a subject number 2368258 and sequence ID

(CP011051.1) states that that subject number produces surfactin.

39

Figure 24. NCBI nBlast sequence alignment of isolate 551 with primer sfp. Identification was 94% to

Bacillus subtilis HJ5, complete genome with a subject number 3627119 and sequence ID (CP007173.1)

states that that subject number produces surfactin.

40

bp 620 623 626 664 672 727 730 NC PC

3,700 bp

3,700 bp

517 524 539 544 551 585 586 615 616

Figure 25. Agarose gel electrophoresis screening for the presence of Bacillus Spp. Amplified with srfAC

(F: 5’- GATCAGGTTCARGAYATGTATTA -3’ and R: 5’- AGCATTTCTGCGTGYGTKCC -3’) in samples of the 16 bacterial isolates that were used, negative control (NC), positive control (PC), and lambda DNA/HINDIII

ladder visualized using a 1% agarose gel with 0.01% SYBR Safe. The expected fragment length is 3,700

bp.

Que ry 4 TGCC-TCCTGJ!..ATCCCGGCC}.A.li.GCTTITJ!.CNTTGA.li.CJ!.A.Z..TCJ!.CGATGA.li..AGTJ!_~.A.li.GG 62 1111 11111111111111111111111111 1111111111111111111111111111

Sbjct 21990 TGCCATCCTGJ!..ATCCCGGCC}.A.~GCTTITACCTTGA.~CJ!.A.~TCACGATGA.li..AGTJ!~.A.~GG 22 049

Que ry 63 CAGCTTGA.~TATC}.A.~TGTCITGA.~GA.~..AGCAT~~..ATGTGATCATGGAOCGGTACGATGT 122 111111111111111111111111111111111111111 11111111111111111111

Sbjct 220SO CAGCTTGA.~TATC}.A.~TGTCITGA.~GA.~..AGCAT~~..ATGT~~TCATGGAOCGGTACGATGT 22109

Que ry 123 ATTTCGTACCGTGTTCATTCACGA.Z.JUL~GTA.Z.JUL~GACCTGTCCA.~GTOGTATTaaaaaa 182 111111111111111111111111111111111111111111111111111111 I I I I I

Sbjct 22110 ATTTCGTACCGTGTTCATTCACGA.Z.JUL~GTA.Z.JUL~GACCTGTCCA.~GTOGTATTG~.A.li.A.~ 22169

Que ry 183 aCGGCAGTTCCATATAGAA~~h.TCGATCTGACACACTTAAOGGGCAGOGAGCAAACAGC 242 111111111111111111111111111111111111111111111111111111111111

Sbjct 22170 ACGGCAGTTCCATATAGJ!..A~~.A.~TCGATCTGACACACTTA.~CGGGCAGOGAGCA.~~CAGC 22229

Que ry 243 CJ!.A.Z...ATCA.~TGAGTACA.Z...A~~..ACAGGATA.~GATCAGGGGTTITGATTTGACGCGGGATAT 302 111111111111111111111111111111111111111111111111111111111111

Sbjct 22230 CJ!.A.Z...ATCA.~TGAGTACA.Z...A~~..ACAGGATA.~GATCAGGGGTTITGATTTGACGCGGGATAT 22289

Que ry 303 TCCGATGCGGGCAGCCATTTTCJ!..AGA~~GCTGAAGA.li..AGCTTTGA.~TGGGTGTGGAGCTA 362 11111111111111111111111111 111111111111111111111111111111111

Sbjct 22290 TCCGATGCGGGCAGCCATTTTCJ!..AGAk~GCTGAAGA.li..AGCTTTGA.~TGGGTGTGGAGCTA 22349

Que ry 363 CCACCACATTATTTTGGAOGGATGGTGCTTCGGCATCGTOGTACAGGATCTATTT}..AGGT 422 111111 11111111111111111111111111111111111111111111111111111

Sbjct 223SO CCACCATATTATTTTGGAOGGATGGTGCTTCGGCATCGTOGTACAGGATCTATTT}..AGGT 22 409

Que ry 423 ATACAATGCTCTGCGCGJ!..AC}.A.li..AGCCGTJ!.CAGCCAGCCCCOCGTCA.Z._~CCGTAT}.A.li.GA 482 11111111111111111111111111111111111 I l l 11111111111111111111

Sbjct 22410 ATACAATGCTCTGCGCGJ!..AC}.A.li..AGCCGTACAGCCTGCCGCOCGTCA.~~CCGTAT}.A.~GA 22 469

Que ry 483 CTACAT}Jl~GTGGCTTGJ!JUL~GCAGGATA.li..ACAAGCATCACTGCGTTACTGGCGCGAGTA 542 111111 11111111111111111111111111111111111111111111111111111

Sbjct 22470 CTACATCA.~GTGGCTTGJ!JUL~GCAGGATA.li..ACAAGCATCACTGCGTTACTGGCGCGAGTA 22529

Que ry S43 TTTAGAGGACTTTGA.~GGAC}.A.~CGACGTTTGOGGAGCJ!Jl~GA.li..AGA.~~CJ!Jlli..AGGACGG 602 111111111111111111111111111111111111111111111111111111111111

Sbjct 22530 TTTAGAGGACTTTGA.~GGAC}.A.~CGACGTTTGOGGAGCJ!Jl~GA.li..AGA.Z._~CJ!Jlli..AGGACGG 22589

Que ry 603 CTATGAGCCGJ!..A.AGAGCTGCTCTTTTCACTGCCGGAU-GCGG~.A.li.CJ!Jl~GGCCTTTACCGA 662 111111111111111111111111111111111111 11111111111111111111111

Sbjct 22590 CTATGAGCCGJ!..A.AGAGCTGCTCTTTTCACTGCCGGAGGCGG~.A.~CJ!Jl~GGCCTTTACCGA 22649

Que ry 663 GCTTGC}JLli..ATOGCAGCATAOCACTTT~..AGTACGGCGCT~~GGCAGTCTGGAGCGTATT 722 111111111111111111111111111111111111111111111111111111111111

Sbjct 226SO GCTTGC}Jlli..ATOGCAGCATAOCACTTT~..AGTACGGCGCT~~GGCAGTCTGGAGCGTATT 22 709

Que ry 723 GATCANCCGCTATCAGCAGTCTGGCGAITTGGCCITCGGTACAGTTGTITCAGGGOGTCC 782 I I I I I 111111111111111111111111111111111111111111111111111111

Sbjct 22710 GATCAGCCGCTATCAGCAGTCTGGCGATTTGGCCTTCGGTACAGTTGTTTCAGGGOGTCC 22 769

Que ry 783 CGCGGA.~..ATCJ!..A.AGGCGTT~~..ACATATGGTCGGGCTGTTTATCJ!..ACGTTGTCCOGAGACG 842 111111111111111111111111111111 11111111111111111 11111111111

Sbjct 22770 CGCGGA.~..ATCJ!..A.AGGCGTT~~..ACATATGGTTGGGCTGTTTATCJ!..ACGTOGTCCOGAGACG 22829

Que ry 843 TGTGA.AGCTGTCTGAli'-GGTJ>~TCJ!.CJ!.TTTA.li.CGGCITGCT~ZI-~GCAlaCTGCJ!.GGAl~CA.~TC 902 111111111111111 11111111111111111111111111111 11111111 I I I I I

Sbjct 22830 TGTGA.AGCTGTCTGAGGGTATCACATTTA.~CGGCTTGCT~~~GCAGCTGCAGGAGCA.~TC 22889

Que ry 903 GCTGCAGTCTGAGCCGCJ!.TC}..ATATGTGCCGCTTTATGA~Z..TCCA.Z...AGOCJ!.G~i'C-GATCA 961 1111111111111111111111111111111111111111111111111111 I I I I I I

Sbjct 22890 GCTGCAGTCTGAGCCGCATC}..ATATGTGCCGCTTTATGA~~TCCA.Z...AGOCAGGCTGATCA 22949

Que ry 962 NNCGAk~CTGA 972 111111111

Sbjct 229SO GCCGAk~CTGA 22960

41

Figure 26. NCBI nBlast sequence alignment of the positive control for gene srfAC. Identification was

100% to Bacillus amyloiquefaciens CC178 with a subject number 21990 and sequence ID (CP006845.1)

states that that subject number produces surfactin.

42

Figure 27. NCBI nBlast sequence alignment of isolate 620 with primer srfAC. Identification was 98% to

Bacillus subtilis subsp. Subtilis positive regulator of hxiAB expression srfAC with a subject number 21990

and sequence ID (JQ073775.1) states that that subject number produces surfactin.

43

bp

620 623 626 664 672 727 730 NC PC

201 bp

201 bp

517 524 539 544 551 585 586 615 616

Figure 28. Agarose gel electrophoresis screening for the presence of Bacillus Spp. Amplified with srfAA

(F: 5’- TCGGGACAGGAAGACATCAT -3’ and R: 5’- CCACTCAAACGGATAATCCTGA -3’) in samples of the 16 bacterial isolates that were used, negative control (NC), positive control (PC), and lambda DNA/HINDIII

ladder visualized using a 1% agarose gel with 0.01% SYBR Safe. The expected fragment length is 201 bp.

44

Figure 29. NCBI nBlast sequence alignment of the positive control for gene srfA. Identification was 100%

to Bacillus amyloiquefaciens srfAA with a subject number 12545 and sequence ID (AJ575642.1) states

that that subject number produces surfactin.

Figure 30. NCBI nBlast sequence alignment of isolate 551 with primer srfAF/R. Identification was 98% to

Bacillus subtilis subsp. Subtilis RO-NN-1, complete genome with a subject number 372482 and sequence

ID (CP002906.1) states that that subject number produces surfactin.

45

bp 517 524 539 544 551 585 586 615 616

290 bp

290 bp

620 623 626 664 672 727 730 PC NC

Figure 31. Agarose gel electrophoresis screening for the presence of Bacillus Spp. Amplified with spaS1

(F: 5’- ATGTCAAAGTTCGATGATTTCGA -3’ and R: 5’- TTATTTAGAGATTTTGCAGTTCA -3’) in samples of the 16 bacterial isolates that were used, negative control (NC), positive control (PC), and lambda

DNA/HINDIII ladder visualized using a 1% agarose gel with 0.01% SYBR Safe. The expected fragment

length is 290 bp.

46

Figure 32. spaS1 NCBI nBlast sequence alignment of the positive control for gene SpaS1 Identification

was 99% to Bacillus subtilis strain 618 subtilin (SpaS) gene, complete cds with a subject number 199 and

sequence ID (DQ452514.1) states that that subject number produces spaS.

Figure 33. spaS11F NCBI nBlast sequence alignment of isolate 616 with primer spaS1F/R. Identification

was 93% to Bacillus subtilis strain 618 subtilin (SpaS) gene, complete cds with a subject number 136 and

sequence ID (DQ452514.1) states that that subject number produces spaS.

47

bp

620 623 626 664 672 727 730 PC NC

566 bp

517 524 539 544 551 585 586 615 616

Figure 34. Agarose gel electrophoresis screening for the presence of Bacillus Spp. Amplified with spaS2

(F: 5’- TGTCATGGTTACAGCGGTATCGGT -3’ and R: 5’- AGTGCAAGGAGTCAGAGCAAGGTGA -3’) in samples of the 16 bacterial isolates that were used, negative control (NC), positive control (PC), and lambda

DNA/HINDIII ladder visualized using a 1% agarose gel with 0.01% SYBR Safe. The expected fragment

length is 566 bp.

48

Figure 35. NCBI nBlast sequence alignment of the positive control for gene SpaS2. Identification was

99% Bacillus subtilis spaB, spaT, spaC, subtilin (spaS), spaI, spaF, spaE, spaG, spaR, spaK, YvaN gene,

complete cds with a subject number 6962 and sequence ID (U09819.1) states that that subject number

produces spaS.

49

Discussion

The goal of this study was to determine if 16 Bacillus samples isolated from the skin of the North

American bullfrog (Lithobates catesbeiana) and California toad (Anaxyrus boreas halophilus) possess

genes that code for enzymes that are involved in known antibiotic biosynthetic pathways. The

antibiotics of interest were flagellin, iturin A, mycosubtilin, translocation-dependent antimicrobial spore

component, subtilin, surfactin, and subtilosin A. Although all of the bacterial isolates tested negative for

primer pairs hagF/R, ituAF/R, mycBF/R, tasAF/R, and spaS2F/R, that amplify genes known to play a role

in the production of flagellin, iturin A, mycrosubtilin, subtilin, and translocation-dependent antimicrobial

spore component (TDA), respectively, primer pairs sfpF/R, srfA-CF/R, srfA-AF/R, sboAF/R, and spaS1F/R,

that amplify genes known to play a role in the production of surfactin, subtilosin A, and subtilin showed

positive results.

Five (551, 615,616,620, and 664) of the 16 sample isolates were positive for the sboA gene,

which is involved in the subtilosin A producing pathway. The presence of this gene in these isolates

suggests the production of that antibiotic is occurring in some cases, but not all because multiple genes

in the subtilosin A pathway must be present for the production of the antibiotic to occur (Stein 2005).

For example, in order for the production of subtilosin A to occur, eight (sboAalbABCDEFG) out of the

nine genes (sboAXalbABCDEFG) must be expressed, with sboA encoding presubtilosin, a 43-amino acid

peptide (Velho et al. 2013, Zheng et al. 2000). For example, previous research used PCR in identifying

the Bacillus gene sboA from Bacillus strains from the intestines of Amazon basin fishes (Velho et al.

2013). This study only targeted one subtilosin A gene (sboA), which is in the subtilosin A producing

pathway, and were successful in detecting this gene in all the Bacillus strains tested (Velho et al. 2013).

However, Velho et al. (2013) also used mass spectrometry, which identifies unknown compounds, and

detected clusters that correspond to subtilosin A peptides showing that the bacteria were producing the

antibiotic from the identified genes. They were not only successful in identifying the sboA gene in

50

Bacillus strains, the gene of interest in this study, but also were able to confirm the presence of the

subtilosin A isoforms through mass spectrometry. The use of the mass spectrometry provides additional

for the presence of the sboA gene and indicates subtilosin A production. Therefore, this study’s findings

suggest that the presence of the sboA gene in the isolates implies that the production of subtilosin A

may be occurring.

The detection of the sfp gene (in isolates 517, 551, and 620), which is involved in the surfactin

producing pathway, lead to the investigation of two additional genes (srfAA and srfAC) that are also

involved in the surfactin producing pathway. Additional genes (srfAA and srfAC) were investigated in this

pathway because previous work (Zepeda 2016) had also investigated the SRFDB3 gene in the surfactin

pathway, yet did not yield positive results, where as in my study there were positive results for sfp gene,

which is also involved in the surfactin pathway. Isolate 620 was positive for all three surfactin genes

tested (sfp, srfAA, and srfAC) and isolates 517 and 551 were positive for two of the surfactin genes

tested (sfp and srfAA). Genes sfp and srfAA had similar results in that more isolates had the srfAA gene

than the sfp gene and all isolates that were positive for sfp gene also tested positive for the srfAA gene.

Hassan et al. (2010), was able to successfully amplify the genes sfp and srfAC, which are the

same genes that were tested in my study, in strains B. subtilis NH-100, B. subtilis NH-160, and Bacillus

sp. NH-217 (Hassan et al. 2010). The main goal of this research was to investigate the ability of

sugarcane plants to suppress red rot disease by identifying the presence of the surfactin producing

genes sfp and srfAC (Hassan et al. 2010). Using liquid chromatography mass spectrometry (LC-MS)

Hassan (2010) was able to determine the presence of the antibiotic surfactin. This, therefore, suggests

that the presence of sfp and srfAC genes in the isolates tested in my study means that the production of

surfactin may be occurring.

51

Mora et al. (2011), studied the distribution of six antimicrobial peptide (AMP) genes (srfAA,

bacA, fenD, bmyB, spaS and ituC) in Bacillus spp. from different plant environments in a Mediterranean

land area in Spain and were successful in identifying genes srfAA, bacA, fenD, and bmyB in 184 isolates.

The results from Mora et al. (2011) were similar to my study, in that they were able to successfully

amplify the srfAA gene, to the ones found in Mora et al. (2011) study while also investigating how the

presence of this gene effects plant survival. The previous study (Mora et al. 2011) was similar to my

study in that it was also investigating the presence of the srfAA gene in order to determine if the

presence of the gene results in subtilosin A production and assisting in plant survival.

The detection of the spaS gene (isolate 616) using primer pair spaS1F/R lead to further

investigation of the spaS gene using primer pair spaS2F/R which amplified the end of the spaC gene and

the whole spaS gene. Unlike subtilosin A, the production of subtilin requires the expression of all ten

genes spaBTCSIFEGRK (Velho et al. 2013). Both primers tested for the spaS gene but each amplified

different parts of the gene. Isolate 616 tested positive for spaS1F/R but all isolates tested negative for

spaS2F/R. Possibly meaning isolate 616 has part of the spaS gene that was detected by spaS1 but

something must be occurring that is preventing the detection of spaS2F/R. The absence of detection

may be due to an insertion mutation that has been described in the pathway of surfactin (Nakano et al.

1991).

Surfactin production is dependent on an intact operon, the 5’ end of the operon is responsible

for sporulation, competence, and surfactin production while the 3’ end is only responsible for

sporulation and surfactin production (Nakano et al. 1991). Similar results to subtilin (spaS gene) were

found in Nakano et al. (1988), with bacteria having some of the genes known to be involved in the

surfactin pathway while lacking other known genes involved in the same pathway. An insertion

mutation was found to be responsible for the absence of the other known genes resulting in an inability

to produce surfactin. Nakano et al. (1991) found that the Tn917 transposon insertion mutation is

52

responsible for the disruption of synthesis, sporulation, and competence of surfactin by inserting itself

into the surfactin operon. They tested this process by introducing the Tn917 mutation into a surfactin

producing Bacillus strain (OKB105) via transformation. Transformed bacteria were assayed on blood

agar and were negative for the production of surfactin. Sequencing of the sfp locus of transformed

bacteria showed that the transposon had inserted itself into the surfactin producing pathway causing a

disruption. This mutation causes a blocking of genes within the surfactin operon, which disrupts

expression. A disruption mutation, such as that caused by Tn917, in any part of these genes would cause

complete inhibition of surfactin production. The insertion of this transposon may also cause some genes

to be undetected via PCR. This may also explain why some parts of the gene responsible for production

of surfactin, subtilin, and subtilosin A were identified while others in the same pathway were not.

The presence of the genes tested usually implies that the production of that antibiotic is

occurring, but in some cases multiple genes must be present for the production of the antibiotic to

occur (Stein 2005) and for most antibiotics there are multiple genes involved in the production of the

antibiotic. Although these primer pairs hagF/R, ituAF/R, mycBF/R, tasAF/R, and spasF/R did not

successfully amplify the target genes, other genes in the same pathway may still allow for the

production of the antibiotic. For example, many isolates tested negative for sboA but the isolates might

have other genes that are also involved in the subtilosin A pathway that this study did not test for. This

may be true for other antibiotics such as surfactin and iturin, which have four genes that are involved.

Other examples of pathways with multiple genes involved may include mycosubtilin (3), Subtilin (10),

and subtilosin A (7) (Stein 2005). In other words, although the isolates tested in this study were negative

for some the genes tested that does not mean the antibiotic is not being produced because other genes

that are involved in that pathway may be present but not tested for in this study.

The isolates tested in this study were collected from frogs and toads in the Bakersfield,

California area (Szick et al. 201X). All isolates (544, 585, 586, 626, and 672) from frogs 2 and 5 and toads

53

2 and 4 (Table 5) were negative for all genes tested including those done by Zepeda (2016), which

means that bacterial isolates collected from these frogs and toads did not contain the tested genes, but

other bacteria present on the skin of the frogs and toads, not selected for this study, may contain the

genes of interest in the antibiotics tested in this study or other antibiotics not tested in this study. Other

Bacillus bacteria that are found on the skin of frogs and toads may contain these genes but were not

tested for in this study because they may have not successfully grown in a lab setting. There is also a

high possibility that the bacteria responsible for producing these antifungal compounds cannot be

isolated using culture dependent methods, as an estimated 99% of bacteria cannot be cultured in a lab

setting (Nichols et al. 2010). Meaning that other techniques must be used in order to investigate the

presence of these bacteria.

One of these techniques is the isolation chip (Ichip), which is made up of hundreds of compact

diffusion chambers and introduced natural nutrients to sample bacteria allowing them to grow in media

similar to their natural environment (Nichols et al. 2010). Ling et al. (2015) were successful in identifying

the novel antibiotic teixobactin, which inhibits cell wall synthesis, from previously uncultivable bacteria

using this technique. Another technique, denaturing gradient gel electrophoresis (DGGE), identifies

differences in GC-content in DNA allowing for the identification of individual species (Lauer et al. 2008).

This may have been useful in this study by allowing for more specific identification of a broader range of

cutaneous bacteria. Also, the cutaneous bacteria may have contained genes are known to be involved in

other antibiotic producing pathways.

Another reason for the absence of these genes in bacteria may be due to resistance originating

from cutaneous peptides found on the skin of amphibians. Simmaco et al. (1998) found that glands on

amphibian skin produce biogenic amines, complex alkaloids, and peptides, which are stored as granules

in the lumen of the skin and upon stimulation, are released. These compounds not only play a role in

defense against microorganisms and predators but also in physiological functions of the skin (Simmaco

54

et al. 1998). This means that the frogs and toads may not necessarily be producing the antibiotics

examined but are still capable of fighting off pathogens via these biologically active compounds. The

frogs and toads are producing this on their own and not necessarily fighting off pathogens via bacteria

present on the skin. This idea is supported by the isolates ability to inhibit the growth of some fungal

pathogens and this was determined by challenge assays (Szick et al. 201X).

To determine if the isolates collected exhibited inhibition of fungal growth, the isolates were

challenged against environmental fungi, an amphibian pathogen, and human pathogens using challenge

assays. The 64 original isolates tested against five fungal human pathogens (Szick et al. 201X): Candida

albicans, which causes oral and genital infections, Microsporum gypseum, which causes ringworms,

Cryptococcus neoformans, which causes pneumonia-like infection, Enterococcus faecalis, which causes

septicemia and endocarditis, and Trichophyton mentagrophytes, which causes scalp lesions and skin

inflammation and an amphibian pathogen Basidiobolus ranarum. They were also tested against five

environment fungi: Eupenicillium iavicum, Aspergillus, sp. [A], Aspergillus sp. [B], Cochliobolus sp., and

Galatomyces. Even though most of the Bacillus isolates inhibited the growth of three or fewer

environmental fungi, some of the isolates inhibited the growth of four of the five human pathogens

(Table 5). The 16 isolates used in this study were previously determined to inhibit the growth of at least

one environmental fungus. Zepeda (2016) who also used these isolates did get positive results for genes

orf2 and zmaR, which are involved in Zwittermicin A production, in isolate 664 but got negative results

for genes (bamC, fend, ituD, and srfDB3) known to be involved in the production of the antibiotics

(bacillomycin D, fengycin, iturin A, and surfactin). Considering these isolates were negative for genes

(hag, ituA, mycB, and tasA) known to be involved in the production of the antibiotics (Iturin,

mycosubtilin, flagellin, and Tas A) investigated in this study and those done by Zepeda (2016) then they

could be producing an antibiotic that was not tested for in this study or an unidentified antibiotic that

has yet to be discovered. This idea is supported by Velho et al. (2013) by using mass spectra it revealed

55

that some peaks did not match any known antimicrobial peptides. Therefore, there must be something

else that is being produced that has yet to be discovered.

Significance

Fungal infections are a problem worldwide and have had a huge impact on economic losses,

food safety, human/animal diseases, and agriculture (Zhao et al. 2016). The prevention and

treatment of this problem is not only important for agriculture and food industry but can also have a

huge impact on medicine (Zhao et al. 2016). Studies have shown that plants that are grown in soils

that are drenched in Bacillus subtillis survived infections better than those soils that did not contain

Bacillus subtilis (Yang et al. 2015). Also, many strains within the genus of bacteria Bacillus have been

reported to control plant diseases by helping plants survive infections (Mora et al. 2011). The key

factors that play a role in why Bacillus are successful in controlling plant disease are their safety,

distribution in diverse habitats, and ability to survive conditions (Mora et al. 2011). With all these

benefits my goal with the identification of these antibiotic synthesis pathways is to be able to better

assist in agriculture and medicine to prevent the spread of more infectious diseases.

Antibiotics today are the main treatment for bacterial and fungal infections and with our

findings and future research there is a potential for discovering a better source of treatment than

chemically synthesized antibiotics. Antibiotic resistance is evolving faster than the discovery of new

antibiotics is occurring (Ling et al. 2015). This means that bacterial and fungal infections are

becoming more resistant faster than the production of chemically synthesized antibiotics. The

discovery of naturally occurring antibiotics will not only in treating current infections, but possibly

preventing the spread of more infections. Using naturally occurring antibiotics can also decrease the

harmful side effects of chemically synthesized antibiotics. Chemically synthesized antibiotics have

been shown to cause DNA methylation, which in turn causes cancer (Schmitt et al. 1997). Naturally

occurring antibiotics are less harmful than those synthesized in the lab. Therefore, our findings will

56

benefit humans/animals, and plants. Therefore, further understanding how some frogs and toads are

able to fight off Bd will assist in further research on fighting off Bd and preventing the spread of it.

Antibiotics today are a key tool in treating infections, but the misuse has caused a great deal

of harm in the ability to fight off future infections with current antibiotics. With the identification of

genes that are involved in the antibiotic biosynthetic pathways within certain bacteria, I hope to

gain a better understanding of these genes and in turn help future research on the synthesis of a

stronger naturally occurring antibiotics.

Conclusion and Future Projects

The Bacillus isolated from the California toad and North American bullfrog contained genes that

are known to be involved in antibiotic biosynthetic pathways of surfactin, subtilin, and subtilosin A.

These findings are key tools that can be used to further investigate natural antibiotics that may prevent

the spread of diseases and prevent the extinction of animals. For future research more investigation can

aid in amplifying multiple genes that are known to be involved in the production of certain antibiotics

and determine the presence of these genes in isolates. For example, focusing on two antibiotics and

target all the genes known to be involved in the production of the antibiotic and then run more research

on the isolates ability to inhibit various types of fungi after detecting the presence of all the genes. Also,

another route that can be taken would be to test the isolates on human pathogens and determine their

level of inhibition. Future research can also target genes and antibiotics that were not tested in this

study or in identifying antibiotics that have not being identified using iCHIP or mass spectrometry as

done in recent discoveries of antibiotics. With the current problem of antibiotic resistance and misuse of

antibiotics a more natural source of antibiotics might be the resource needed to prevent more

resistance amongst bacteria.

57

Table 5. Results of Challenge assays (in prep, Szick et al. 201X) for Bacillus spp against environmental fungi (Eupenicillium iavicum,

Aspergillus, sp. [A], Aspergillus sp. [B], Cochliobolus sp., and Galatomyces., an amphibian pathogen (Basidiobolus ranarum) and human

pathogens (Trichophyton mentagrophytes, Enterococcus faecalis, Microsporum gypseum, Cryptococcus neoformans and Candida albica.

Amphibian

FROG 1

Original

ID

517

524

539

Ej

X

X

Environmental Fungi

A sp. A sp. C sp

X

X

X X

Gg

X

Amphibian

Pathogen

Br

X

X

X

Tm

X

X

Human Pathogens

Ef Mg Cn

X X

X X X

X X

Ca

X

FROG 2 544 X X X X X X

FROG 3 551 X X X X X X X

FROG 5 585

586

X

X

X

X

X

X

X

X

X

X

X X

TOAD 1 615

616

620

623

X

X

X

X

X

X

X

X

X

X

X

X

X

X

X

X

X

X

X

X

X

TOAD 2 626 X X

TOAD 3 664 X X X X

TOAD 4 672 X X X X X

TOAD 5 727

730 X

X

X X X

X

X

X

X X

X

X X

X

X

58

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