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농학석사학위논문

해양방선균 Streptomyces bacillaris 유래 항균활성

물질 연구

A Study on the Antibacterial Secondary Metabolites from

Marine-derived Streptomyces bacillaris

2019년 2월

서울대학교 대학원

농생명공학부 응용생명화학전공

배 수 현

A Dissertation for the Degree of Master of Science

A Studies on the Antibacterial Secondary Metabolites

from Marine-derived Streptomyces bacillaris

February 2019

Suhyun Bae

Applied Life Chemistry Major

Department of Agricultural Biotechnology

Seoul National University

해양방선균 Streptomyces bacillaris 유래

항균활성물질 연구

A Study on the Antibacterial Secondary Metabolites

from Marine-derived Streptomyces bacillaris

지도교수 오 기 봉

이 논문을 농학석사학위논문으로 제출함

2018 년 12 월

서울대학교 대학원

농생명공학부 응용생명화학전공

배 수 현

배수현의 석사학위논문을 인준함

2019 년 2 월

위 원 장 이 상 기 (인)

부 위 원 장 배 의 영 (인)

위 원 오 기 봉 (인)

i

Abstract

The actinomycetes from diverse marine environments are very prolific sources of

structurally unique and biologically active metabolites. The decline in the efficiency

of discovering antibiotics from terrestrial microbes has increased natural products

chemists’ interest in investigating the chemistry of marine microorganisms,

particularly as antibiotic resistance emerges as a significant threat to human health.

In this study, 406 marine-derived actinomycetes provided by Korea Institute of

Ocean Science & Technology were screened to search for antimicrobial compounds.

Among them, the actinomycete strain 38C, which showed potent antibacterial

activity, was further examined. This strain was identified as Strptomyces bacillaris

by 16s rRNA analysis. The bacterium was cultured in GTYB50 at 28℃ for 5 days

and the culture broth was extracted with organic solvents. Bioassay-guided

separation of the crude extract using various chromatographic techniques yielded an

active compound. On the basis of the results of combined spectroscopic data, this

compound was determined to be medermycin, a pyranonaphthoquinone. The

antimicrobial activity of medermycin was evaluated against various pathogenic

bacteria and fungi. This compound exhibited potent inhibitory activities against

Gram-positive and Gram-negative bacteria including MRSA.

Keywords: marine actinomycetes, Strptomyces bacillaris, secondary metabolites,

chemical structure, antibacterial activity

Student Number: 2017-23390

ii

Contents

Abstract ...................................................................................................................................... i

Contents..................................................................................................................................... ii

List of Figures ......................................................................................................................... iv

List of Tables ........................................................................................................................... v

List of Abbreviations ............................................................................................................ vi

Introduction ............................................................................................................................. 1

Materials and Methods ........................................................................................................ 6

Screening of antimicrobial activity from marine-derived actinomycetes ....... 6

Identification of the actinomycete strain 38C ................................................... 7

Culture condition of the strain 38C ................................................................... 7

Isolation of antibacterial compound .................................................................. 8

Structure elucidation ........................................................................................... 9

Antibacterial activity assay ................................................................................. 9

Antifungal activity assay ................................................................................... 10

Paper disc assay ................................................................................................. 11

Time-kill assay ................................................................................................... 11

Membrane potential assay ................................................................................ 12

Effect of culture medium on the production of antimicrobial metabolites .. 12

Results ..................................................................................................................................... 14

Identification of 38C .......................................................................................... 14

Culture condition of S. bacillaris ...................................................................... 18

iii

Isolation of antibacterial compound P2 ........................................................... 23

Structure elucidation ......................................................................................... 29

Antimicrobial activity of compound ................................................................ 34

Time-kill assay ................................................................................................... 40

Membrane potential assay ................................................................................ 44

Effect of culture medium on the production of antimicrobial metabolites .. 48

Discussion .............................................................................................................................. 53

Supplementary Materials ................................................................................................. 55

Reference ............................................................................................................................... 82

Abstract in Korean ............................................................................................................. 87

iv

List of Figures

Figure 1. Phylogenetic tree of Streptomyces bacillaris ........................................... 16

Figure 2. Antibacterial activities of S. bacillaris culture liquid from various

medium .................................................................................................... 20

Figure 3. Isolation procedure of antibacterial compound ........................................ 24

Figure 4. Partitioning 38C EtOAc extract on HPLC chromatogram ....................... 26

Figure 5. Structure of compound P2 ....................................................................... 30

Figure 6. Paper disc assay against S. aureus and C. albicans ................................. 38

Figure 7. Time-kill assay ......................................................................................... 42

Figure 8. Membrane potential assay ....................................................................... 46

Figure 9. Agar diffusion assay against S. aureus with various media ..................... 50

v

List of Tables

Table 1. Antibacterial activities of organic solvent extraction from S. bacillaris

culture medium ........................................................................................ 22

Table 2. MIC value of each part of chromatogram ................................................. 28

Table 3. 13C NMR assignments for compound P2 .................................................. 32

Table 4. 1H NMR assignments for compound P2 ................................................... 33

Table 5. Antimicrobial activities of compound P2 against Gram-positive bacteria,

Gram- negative bacteria, and fungi ......................................................... 35

Table 6. Antibacterial activities of compound P2 against MSSA and MRSA ........ 36

Table 7. MIC assay with various media .................................................................. 52

vi

List of Abbreviations

ACN acetonitrile

Aq. aqueous

Amp ampicillin

ATCC American Type Culture Collection

BLAST basic local alignment search tool

CCARM Culture Collection of Antimicrobial Resistant Microbes

CDCP Center for Disease Control and Prevention

CFU colony forming unit

COSY correlation spectroscopy

DMSO dimethyl sulfoxide

DW distilled water

EtOAc ethyl acetate

GTYB glucose, tryptone, yeast extract, beef extract mixed medium

GTYB50 glucose, tryptone, yeast extract, beef extract, sea salt (50% of sea)

mixed medium

HPLC high performance liquid chromatography

HMBC heteronuclear multiple bond correlation

HSQC heteronuclear single quantum coherence

KIOST Korea Institute of Ocean Science & Technology

MeOH methanol

vii

MBC Minimum Bactericidal concentration

MHB Muller Hinton Broth

MIC minimal inhibitory concentration

MRSA methicillin-resisrance Staphylococcus aureus

MSSA methicillin-sensitive Staphylococcus aureus

MS mass spectrometry

NBRC National Institute of Technology and Evaluation Biological Resource

n-Hx normal-hexane

NCBI national center for biotechnology information

NMR nuclear magnetic resonance

O.D.600 optical density at wavelength 600 nm

PNQ pyranonaphthoquinone

Tet tetracycline

TFA trifluoroacetic acid

RI refractive index

1

Introduction

The term antibiotic, which means “against life” from ancient Greek roots, is a type

of substance active against microbes and also refer to a compound killing or

inhibiting the growth of bacteria (Waksman, 1947).

Antibiotics are commonly classified by their chemical structure, mechanism of

action, and spectrum of activity. The targets of antibiotics are the bacterial cell wall

and envelope (penicillins and cephalosporins), protein synthesis inhibition

(macrolides, lincosamides, and tetracyclines), and nucleic acid inhibition

(Rifampicin) (Finberg et al., 2004). The activity spectrum of antibiotics consist of

narrow-spectrum (Clidamycin) and broad-spectrum (fluoroquinolone). Narrow-

spectrum antibiotics is target specific types of bacteria like gram-positive or gram-

negative. Broad-spectrum is target a wide range of bacteria (Buckel et al., 2017). The

chemical structure of antibiotics is categorized by β-lactams (penicillins,

cephalosporins, carbapenems, monobactams), macrolides (erythromycin),

polyketides (tetracyclines), aminoglycosides (streptomycin), glycopeptides

(vancomycin), lipopeptides (daptomycin), quinolones (ciprofloxacin),

oxazolidinones (linezolid), and sulphonamides (Frank and Tacconelli, 2009; Van

Hoek et al., 2011). In addition, the last three groups, quinolones, oxazolidinones,

sulphonamides, are not from nature while the others are from natural products

(Hughes and Fenical, 2010).

Secondary metabolites, including antibiotics, are organic compounds produced

by bacteria, fungi, or plants in nature. These compounds are not directly involved in

normal growth, development, or reproduction of organism directly. Absence of

secondary metabolites does not result in immediumte death like primary metabolites,

2

but in long-term impairment of the organism’s survivability (Pichersky and Gang,

2000). Almost 22,500 bioactive secondary metabolites from natural source have

been reported. Out of these total bioactive secondary metabolites, 45% (10,100) are

produced by actinomycetes in which 7,630 are from strptomycetes and 2,470 are

from rare-actinomycetes (Selvameenal et al., 2009).

Actinomycetes are Gram-positive bacteria that have a DNA with a high content

of guanine-cytosine (69 to 73 mol %). Some are beneficial sources of antibiotics

including important antimicrobial drug classes such as β-lactams, tetracyclines,

macrolides, glycopeptides, and aminoglycosides (Sanglier et al., 1993; Genilloud,

2017). Actinomycetes are being isolated and identified world-wide in the natural

habitats such as soil from various ecological units, marine water, snad, pollen grain,

alkaline waters etc. Among the genera of actinomycetes, the genus of Streptomyces

has the largest number of species and varieties, which differ in their morphology,

physiology, and biochemical activities (Taddei et al., 2006). Streptomyces have

shown the ability to make antibacterial, antifungal, insecticidal, antitumor, antiviral

herbicidal and plant growth promoting compounds (Sacramento et al., 2004;

Prabavathy et al., 2006; Sousa et al., 2008; Hong et al., 2009; Ramesh et al., 2009;

Pimentel-Elardo et al., 2010). Marine-derived Streptomyces occur in various

biological source such as fish, sponge, seaweed beside seawater and sediments

(Feling et al., 2003; Subramani and Aalbersberg, 2012). Isolation of Streptomyces

from marine sediments may be valuable for the production of new antibiotics.

The soil is a natural reservoir for microorganisms and their metabolites include

antimicrobial products (Dancer, 2004). Billions of microbes are compete to survive

in the soil. Early scientist observed the antagonism in the soil flora and speculated

that the existence of antimicrobial metabolites is key to its survival (Mahajan and

3

Balachandran, 2012). The search for novel antibacterial metabolites took a new

direction to expand its searching pool to plants and animals in the sea. Now, as the

result of this research, the study of marine natural products is recognized as both a

significant resource for new drug discovery and an integral component of natural

products (Jensen and Fenical, 1994).

Since late 1980s, the number of novel bioactive compounds isolated from

terrestrial microorganisms steadily decrease. In result, researchers have been focused

on microorganisms in unusual environments for novel compounds (Ramesh et al.,

2009). Because environmental conditions of the sea is different from terrestrial

conditions (Kijjoa and Sawangwong, 2004) marine-derived actinomycetes may have

different characteristics and produce novel bioactive compound (Ellaiah and Reddy,

1987; Ramesh et al., 2009) The discovery of promising drug leads has revealed that

marine actinomycetes produce novel biologically active secondary metabolites and

have potential to be developed as therapeutic agents (Feling et al., 2003; Maldonado

et al., 2005; Asolkar et al., 2010).

After discovery of penicillin and streptomycin in 1928 and in 1943 respectively,

the golden age of antibiotics began. Shortly thereafter, penicillin-resistant bacteria

emerged and spread rapidly (Spellberg and Gilbert, 2014). Strains of MRSA first

appeared in hospitals after commonly prescribing antibiotics as an infection

treatment in the 1960s and now MRSA is the major causative agent of hospital and

community acquired infections. Today, infectious diseases are a major cause of

deaths world-wide, with around 13.3 million constituting for 25% of all deaths

(Selvameenal et al., 2009). In 1974, only 2% of Staphylococcus infections were

drug-resistance but in 2004, 63% of Staphylococcus were. The Center for Disease

Control and Prevention (CDCP) has reported that Staphylococcus infections kill

4

more people in the U.S.A than AIDS (Mahajan and Balachandran, 2012).

Novel antibiotics can enhance the therapeutics pipeline by overcoming

antibiotic resistance. Finding lead compounds can overcome antibacterial-resistance

because they will not be cross-resistant with existing drug.

For identifying antibacterial ability, two groups of bacteria were used, Gram-

positive and Gram-negative. In the group of Gram-positive bacteria, Staphylococcus

aureus, Bacillus subtilis, Enterococcus faecium, and Enterococcus faecalis were

used. S. aureus is one of the important bacterial pathogen in medical (Kali, 2015)

because S. aureus is a pathogen to both bacterium and human and approximately 30%

of humans are colonized with S. aureus (Wertheim et al., 2005). Initially, most of S.

aureus were sensitive to penicillin but, many infections became resistant to penicillin

and methicillin in the 1950s (Chambers and Deleo, 2009). B. subtilis (B. subtilis)

spores can survive in extreme heat and some strains are responsible for causing

ropiness, a stichy, stringy consistency caused by polysaccharides, in spoiled bread

dough (Pepe et al., 2003). Enterococci are Gram-positive bacteria that are commonly

found and E. faecium and E. faecalis are two most characterized members. These

two are also the main cause of enterococci infections in human (Willems and Van

Schaik, 2009). In the group of Gram-negative bacterial pathogens, Salmonella

enterica, Klebsiella pneumoniae, Escherichia coli, and Proteus hauseri are used. S.

entrica is an intracellular bacterial pathogen that causes gastroenteritis typically

acquired by ingestion of contaminated food or water (Flores-Díaz et al., 2015). K.

pneumoniae is a pathogen that cause severe infections such as septicaemia,

pneumonia, and urinary tract infections (Wu and Li, 2014). E. coli can cause

gastroenteritis, urinary tract infections, and neonatal meningitis (Lim et al., 2010). P.

hauseri are commonly responsible for urinary and septic infections (Jacobsen et al.,

5

2008).

In this study, to search for antimicrobial compounds, marine-derived

actinomycetes provided by KIOST were screened for their inhibitory activities

toward S. aureus and K. pneumoniae. Among them, the actinomycete strain 38C,

which showed potent antibacterial activity, was further investigated. This strain was

identified as Streptomyces bacillaris by 16s rRNA analysis. The bacterium was

cultured in GTYB50 medium and the culture broth was extracted with organic

solvents. Bioassay-guided separation of the crude extract using various

chromatographic techniques yielded an active compound. On the basis of the results

of combined spectroscopic data, this compound was determined to be medermycin,

a pyranonaphthoquinone. This compound exhibited significant inhibitory activities

against Gram-positive and Gram-negative bacteria including MRSA.

6

Materials and Methods

Screening of antimicrobial activity from marine-derived actinomycetes

The 406 of marine-derived actinomycetes were provided by Korea Institute of Ocean

Science & Technology (KIOST). To preserve them, streaking all of sample and

making slant and stock (15% glycerol) for clear isolation. For these experiments,

seed medium was used which is consisted of 5 g of glucose, 10 g of starch, 5 g of

peptone, 2 g of yeast extract, 17 g of sea salts dissolved in 1 L of distilled water.

To investigate antimicrobial activity from marine-derived actinomycetes, two

different type of culture were applied, stationary liquid culture and solid culture. At

the first, for liquid culture, culture medium (Hu and Macmillan) was used which is

consisted of 5 g of glucose, 10 g of starch, 5 g of peptone, 5 g of yeast extract, 17 g

of sea salts dissolved in 1 L of distilled water. The strain 38C was cultured in 100

mL of liquid medium at 30℃ with stationary. After incubating the bacterial culture

for 7 days, the culture was filtered by filter paper (300 nm, qualitative, Advantec,

Japan) to separate the mycelia from the fermented medium. The filtrate was extracted

with Ethyl Acetate (EtOAc) as same volumes for three times. The EtOAc

layer was evaporated in vacuo at 34ºC and then the residue was dissolved in DMSO

and stored at -20’C. Three different solid culture were progressed to compare

antimicrobial activities: (1) culture medium (5 g of glucose, 10 g of starch, 5 g of

peptone, 5 g of yeast extract, 17 g of sea salts, and 20 g of agar dissolved in 1 L of

distilled water) (Hu and Macmillan); (2) GTYB50 (10 g of glucose, 2 g of tryptone,

1 g of yeast extract, 1 g of beef extract, 17 g of sea salts, and 20 g of agar dissolved

in 1 L of distilled water) (Shin et al., 2003); (3) YPM consisted of 4 g of mannitol, 2

g of peptone, 2 g of yeast extract, 17 g of sea salts, and 20 g of agar dissolved in 1 L

7

of distilled water (Kim et al., 2012). All medium were sterilized by autoclaving at

121℃ for 20 minutes. To compare antibacterial activity, top agar assay method was

used. One ul of the actimycetes spore solution was spotted on the three of different

type media and covered by top agar which contain S. aureus. The plate was cultured

at 35℃ for 2 days and inhibition zone was observed.

Identification of the actinomycete strain 38C

The strain 38C was phylogenetically identified as a Strptomyces bacillaris (100%

identity) based on the 500 bp of 16S rRNA gene sequence analysis. Identification

was performed by Charles River Korea.

Culture condition of the strain 38C

To select proper medium and type of culture, two category, four type of culture types

were used: (1) the volume of culture: 100 mL culture volume was compare with 500

mL culture volume; (2) sea salt addition to GTYB medium GTYB50 medium is

GTYB medium added 17 g of sea salts. The incubation of strain 38C was carried out

by transfer of 25 mL of seed culture to 100 mL or 500 mL of same medium and

incubation at 30℃ for 3 days and 7 days, 100 mL culture volume and 500mL culture

volume, respectively. All of the culture were with shacking at 130 rpm. After

incubation, the culture was filtered by filter paper (300 nm, qualitative, Advantec,

Japan) to separate the mycelia from the fermented medium. The filterate was

extracted with hexane (Hx), ethyl acetate (EtOAc), n-buthanol (n-BuOH) as same

volumes for three times. All of the extract was concentrted in vacuo at 34’C and then

the residue was dissolved in DMSO and stored at -20’C.

8

Isolation of antibacterial compound

1) Cultivation of the strain 38C

The strain 38C was cultured in GTYB 50 medium at 30℃ with shacking at 130 rpm

for 5 days. The culture was filtered filtered by filter paper (300 nm, qualitative,

Advantec, Japan). The dried residue was extracted with MeOH for desalting.

2) Organic solvent partitioning of 38C extract

The dried MeOH extract was dissolved in water again and liquid-liquid partitioning

was conducted between water and organic solvent (Hx, EtOAc, n-BuOH) serially.

Each solvent was partitioning as same volume for tree times and then each solvent

layer was dried under reduced pressure.

3) High performance liquid chromatography (HPLC)

The EtOAc fraction was collected and the antibacterial compound was separated by

HPLC. HPLC were conducted on a TRILUTION LC control software with a 321

pump, a UV/VIS-151 detector (Gilson, Middleton, WI, USA). Semi-preparative

column (Agilent ZORBAX Eclips Plus C18 4.6 × 250 mm) with guard column was

used. Sample was dissolved in MeOH (HPLC grade). Mobile phase was mixed of

acetonitrile (ACN) and water with 0.1% trifluoroacetic acid (TFA). Flow rate was 2

mL/min. Gradient program was 20% ACN to 100% ACN in 40 min running time

and UV wavelength 254 nm was used.

9

Structure elucidation

1D and 2D NMR spectra (1H, 13C, 1H-1H COSY, HSQC, and HMBC) were recorded

in Methanol-d4 solution on Bruker AVANCE 600 spectrometer (Bruker BioSpin Ltd.,

Germany). Both proton and carbon NMR spectra were measured at 600 MHz,

Cryoprobe (z-gradient, 5mm TXI). NMR spectra were provided by NICEM for

Seoul national university, Seoul, Korea.

Antibacterial activity assay

The following 6 microorganisms, obtained from the stock culture collection at

American Type Culture Collection (ATCC) (Rockville, MD, U.S.A), National

Institute of Technology and Evaluation Biological Resource Center (NBRC) (Japan),

and Culture Collection of Antimicrobial Resistant Microbes (CCARM) (Korea)

were used for antibacterial activity assay: S. aureus ATCC 25923, B. subtilis ATCC

6633, E. faecium ATCC 19434, E. faecalis ATCC 19433, S. entrica ATCC 14028, K.

pneumoniae ATCC 10031, E. coli ATCC 25922, and P. hauseri NBRC 3851. In

addition, two type of S. aureus was used: (1) methicillin sensitive S. aureus CCARM

0027, CCARM 0204, CCARM 0205. (2) methicillin resistant S. aureus CCARM

3640, CCARM 3089, CCARM 3090, CCARM 3634, CCARM 3635, ATCC 43300,

ATTCC 700787, and ATTCC 700788. Bacteria were grown overnight in MHB

(Muller Hinton Broth) at 37℃. Antibacterial activity was determined by MIC

(Minimum inhibitory concentration) assay, which is determined as the lowest

concentration of test compounds that inhibits bacterial growth. The two-fold

microtiter broth serial dilution method (Peloquin et al., 1989). Dilution of compound

dissolved in DMSO were added to each well of 96-well microtiter plate containing

fixed volume of MHB (MB cell). The concentration of compounds ranges from 64

10

to 0.125 ug/mL. Each well was inoculated with an overnight culture of bacteria (5 x

105 CFU/mL), and incubated at 37’C for 16 h. Ampicillin (Amp) and tetracycline

(Tet) were used as positive control compounds. To determine MBC (Minimal

bactericidal concentration), 100 µL culture from each well from 96-well microtiter

plate were withdrawn for determination of bacterial counts. Colony counts were

determined by plating each diluted sample onto MHB agar (MB cell) and incubated

at 37℃ to confirm colony counts.

Antifungal activity assay

Aspergillus fumigates HIC 6094, Trichophyton rubum NBRC 9185, Trichophyton

mentagrophytes IFM 40996, and Candida albicans ATCC 10231 were used for

antifungal activity assay. A. fumigates, T. rubum, and T. mentagrophytes were grown

in potato dextrose agar (PDA, Acumedium Manufacturers, Inc., Maryland) medium

at 30℃ for 2 weeks. C. albicans was grown in YPD (1% yeast extract, 2% peptone,

and 2% glucose) medium at 30℃ for 12 h. The antifungal activities of compound

was determined by following broth dilution method M27-A2, which was proposed

by the National Committee for Clinical Laboratory Standards (Pfaller et al., 2000).

Each compound dissolved in DMSO was added to each well of 96-well microtiter

plate containing fixed volume of RPMI 1640 (Sigma) to prepare serial two-fold

dilution. The concentration of compounds ranges from 64 to 0.125 μg/mL. In the

case of A. fumigates (0.4-5 × 104 spore/mL), T. rubum, and T. mentagrophytes (1-

3 × 103 spore/mL) were inoculated in 100 microliters of the broth. In the case of C.

11

albicans, about 0.5-2.5 × 103 spore/mL was inoculated in 100 microliters of the

broth. The MIC values were determined after incubation at 30℃ for 48 hours.

Amphotericin B was used as a positive control.

Paper disc assay

To confirm selective antimicrobial activity between prokaryotic and eukaryotic,

paper disc assay was conducted. S. aureus were spread on MHB agar and C. albicans

were spread on YPD agar. Paper discs are autoclaved and allowed to dry before use

(De Beer and Sherwood, 1945). In the middle of agar plate, put the paper disc and

drop compound solution. After incubating 16~20 h for S. aureus and 24 h for C.

albicans, inhibition zone was observed.

Time-kill assay

The fresh S. aureus, S. aureus MRSA 700787 colony from overnight growth were

adjusted and diluted cell density to 106 CFU/mL. Each 5mL culture was placed in

test tubes with 8x MIC concentrations of the compound. The test tubes were

incubated in shacking incubator at 37℃, and 100 µL culture were withdrawn for

determination of bacterial counts at 0, 2, 4, 6, 9, 12, and 24 h. Colony counts were

determined by plating each diluted sample onto MHB agar (MB cell) and incubated

at 37℃ to confirm colony counts (Tsuji et al., 2008).

A control test as performed without antibiotics. The procedure was performed in

three independent experiments and a graph of the log CFU/mL was plotted against

12

time (Appiah et al., 2017).

Membrane potential assay

Membrane potential of S. aureus cells was measured by using a fluorescent dye 3,3-

dipropylthiacarbocyanine (DiSC3(5); Anaspec) based on the previous described

method of Wu and Hancock (24). The DiSC3(5) is a fluorescent probe which can

accumulate and become self-quenched in the polarized cell membrane. The probe

release when cell membrane depolarized, it leads to change its fluorescence. Early

exponential phase S. aureus were collected by centrifugation (5,000 rpm, 10 min),

washed twice and resuspended in buffer A (5 mM HEPES (sigma), 5 mM glucose;

pH 7.2) to an optimal density of 0.05. The fluorescent probe DiSC3(5) was added to

a final concentration of 0.4 uM and KCl to a final concentration of 100 mM. The

mixture was incubated to allow the uptake of the DiSC3(5) probe until there was a

stable reduction in fluorescence and treated with 4x MIC Nisin for positive control

and medermycin. A change of fluorescence were monitored by Fluorescence

spectrometer (FluoroMate FS-2, Sinco) at 622 nm excitation wavelength and 670

nm emission wavelength (Cheng et al., 2014) by NICEM for Seoul national

university, Seoul, Korea.

Effect of culture medium on the production of antimicrobial metabolites

To investigate the effect of culture medium on the production of antimicrobial

metabolites, S. bacillaris was cultured in four different type of medium (YPM, M2,

GTYB, and Gause’s medium) at 28ºC with shacking at 130 rpm: (1) YPM (4 g of

Mannitol, 2 g of Yeast extract, 2 g of Peptone, and 17 g of Sea salt dissolved in 1 L

13

of water) (2) M2 (4 g of Glucose, 10 g of Glycerol, 4 g of Yeast extract, 1 g of Malt

extract, and 17 g of Sea salt dissolved in 1 L of water) (3) GTYB’ (4 g of Glucose,

10 g of Glycerol, 4 g of Starch, 2 g of Tryptone, 1 g of Yeast extract, 1 g of Beaf

extract, and 17 g of Sea salt dissolved in 1 L of water) (4) Gause’s (20 g of Starch, 1

g of KNO3, 0.5 g of MgSO4, 0.01 g of FeSO4 7water, 0.5 g of KH2PO4, and 17 g of

Sea salt dissolved in 1 L of water). After incubating for 9 days, the culture was

filtered concentrated in vacuo, and extracted with MeOH for desalting. The dried

residue was dissolved in water again extracted with EtOAc and then analyzed by

HPLC.

14

Results

Identification of 38C

The strain 38C was phylogenetically identified as a Streptomyces bacillaris (100%

identify) based on the 500 bp of 16S rRNA gene sequence analysis (Charles River

Korea). BLAST search data also revealed that 16S rRNA gene sequence of 38C was

matched with Streptomyces bacillaris perfectly (Figure 1). Streptomyces bacillaris

is a bacterium species which produced the bioactive small molecules. Recently a

novel peptide was discovered which have autophagy inhibitory activity (Hu and

Macmillan, 2012).

15

16

Figure 1. Phylogenetic tree of Streptomyces bacillaris

The strain 38C shows 100% similarity with Streptomyces bacillaris

17

18

Culture condition of S. bacillaris

According to first 406 of marine-derived actinomycetes sreening results, the

inhibition zone is largest with 100 ml scale and GTYB50 (salt-added GTYB medium)

that means GTYB50 medium with 100 ml scale is an appropriate condition to

produce antibacterial compound from S. bacillaris (Figure 2). So, 100 ml scale

GTYB50 was selected and used for second stage cultivation condition screening,

EtOAc extract from GTYB50 exhibited significant antibacterial activity than salt-

free GTYB medium (Table 1). Taken together, 100 ml scale GTYB50 medium is an

appropriate condition to produce antibacterial compound from S. bacillaris. Before

cultivation for 3 days in this condition, seed culture was conducted in 25 ml scale of

GTYB50 medium for 2 days.

19

20

Figure 2. Antibacterial activities of S. bacillaris culture liquid from various

medium

Each medium was cultured at 30℃ with shaking at 130 rpm for 2 weeks after

inoculation of S. bacillaris. Each square are consisted small scale GTYB medium,

small scale GTYB50 medium, large scale GTYB medium, and large scale GTYB50

medium. The largest inhibition zone is with small scale GTYB50 medium.

21

100 ml 500 ml

GTYB GTYB50 GTYB GTYB50

agianst Staphylococcus aureus ATCC 25923

22

Table 1. Antibacterial activities of organic solvent extraction from S. bacillaris

culture medium

Option Organic solvent MIC (μg/ml)

(vs a

S. aureus)

100 ml GTYB

Hexane 8

Ethyl acetate 16

Buthanol 4

100 ml GTYB50

Hexane 64

Ethyl acetate 0.5

Buthanol 2

500 ml

GTYB

Hexane 16

Ethyl acetate 16

Buthanol 8

500 ml GTYB50

Hexane 16

Ethyl acetate 4

Buthanol 32

c

Amp 0.125

aStaphylococcus aureus ATCC 25923,

cAmpicillin was used as positive control

23

Isolation of antibacterial compound P2

The bioactive compound was purified by following procedure (Figure 3)

The EtOAc extraction crude were separated by HPLC. As conducted

chromatographic condition, chromatogram was partitioned as 7 parts, 3 peaks and 4

fractions, and confirmed MIC value against S. aureus respectively (Figure 4 and

Table 2). The bioactive peak was peak 2 (P2) with MIC of 0.25 μg/ml against S.

aureus. The P2 was collected and purified by reverse phase semi-preparative column

descried before using Agilent ZORBAX Eclips Plus C18 4.6 x 250 mm (Figure S1).

24

Figure 3. Isolation procedure of antibacterial compound

The compound P2 was isolated by this procedure. 38C culture liquid was filtrated and

concentrated. After organic solvent extraction, HPLC was conducted.

25

Streptomyces bacillaris

Mycelium Medium

Filtration

Crude Extract

n-Hexane Ethyl Acetate Water

MeOH Extraction

Solvent Partitioning

(87.5 L)

Concentration

Peak

HPLC

ACN, Water (+ 0.1% TFA)

(20 mg)

26

Figure 4. Partitioning 38C EtOAc extract on HPLC chromatogram

Chromatogram was partitioned as 7 parts, 3 peaks and 4 fractions by reverse phase semi-

preparative column descried before using Agilent ZORBAX Eclips Plus C18 4.6 x 250 mm

and ACN and water was using.

27

28

Table 2. MIC value of each part of chromatogram

C is used as positive control

MIC (μg/ml) (vs S. aureus)

Front >64 P1 >64

Middle 1 16 P2 0.25

Middle 2 8 P3 >64

Back >64 cAmp 0.125

29

Structure elucidation

According to MS analysis in positive ion mode at m/z 458.2 for [M + H]+ and in

negative ion mode at m/z 456.1 for [M − H]− (Figure S2), the molecular weight of

compound P2 is 457.

1H (Figure S3) and 13C (Figure S4) NMR data assigned for this compound by 1H-1H

COSY (Figure S5), HMBC (Figure S6), and HSQC (Figure S7) NMR. The 1H NMR

spectrum of compound P2 consisted of many singlets, doublets, and triplets. Proton

resonances at (δH 4.98, 4.72, 5.30, 7.68, 7.92, 4.93, 3.62, 3.42, and 3.53) represent

methines signal. Proton resonances at δH (3.23, 2.43) and (2.34, 1.73) represent

methylenes signal. Proton resonances at δH 12.21 represent alcohol signal. Proton

resonances at δH 1.31, 2.72, 1.49 represent methyl signal. Proton resonances at δH

9.58 represent ammonium signal (Table 4). The 13C NMR data revealed a total of 24

carbon resonances were observed including eight aliphatic carbon (δC 68.8, 66.5,

36.4, 65.7, 17.9, 37.3, 36.4, and 17.9), five tetrahydropyran carbons (δC 70.1, 76.5,

69.3, 65.7, and 29.1), six benzene carbons (δC 156.9, 114.5, 130.4, 118.6, 133.7, and

136.3), two ethylene carbons (δC 134.9, 149.2), one carboxyl carbon (δC 175.2), and

two carbonyl carbons (δC 181.4 and 188.2) (Table 3). Compound P2 was matched

with medermycin (Figure 5).

Medermycin is the antibiotics which known as antitumor agent broadly and have

bioactivity against bacteria. The mechanism of action as antitumor is bioreductive

alkylation mechanism proposed by quinone but antibacterial mechanism is unclear

(Lü et al., 2015) (Salaski et al., 2009).

30

Figure 5. Structure of compound P2

The compound P2 is medermycin. The blue numbers are carbon chemical shifts and the red

numbers are hydrogen chemical shifts. The structure of medermycin can describe amino-C-

glycoside-pyranonaphthoquinone (PNQ) lactone.

31

32

Table 3. 13C NMR assignments for compound P2

Position 13C position 13C 1 65.7 11a 36.4 2 11b 3 66.5 12 175.2 4 68.8 13 17.9 4a 134.9 1' 5 181.4 2' 70.1 5a 130.4 3'a 29.1 6 118.6 3'b 7 133.7 4' 65.7 8 136.3 5' 69.3 9 156.9 6' 76.5 9a 114.5 7' 17.9

9-OH NH+ 10 188.2 NCH3 a 36.4 10a 149.2 NCH3 b 37.3

33

Table 4. 1H NMR assignments for compound P2

Position 1H position 1H 1 4.98 11a 3.23 2 11b 2.43 3 4.72 12 4 5.30 13 1.49 4a 1' 5 2' 4.93 5a 3'a 2.34 6 7.68 3'b 1.73 7 7.92 4' 3.62 8 5' 3.42 9 6' 3.53 9a 7' 1.30

9-OH 12.21 NH+ 9.58 10 NCH3 a 2.76 10a NCH3 b 2.76

34

Antimicrobial activity of compound

The biological activities of the medermycin were evaluated against pathogenic

bacterial strains (Table 5). Medermycin exhibited potent antibacterial activities

against both of Gram-positive and Gram-negative bacteria with MIC averages

ranging between 0.0625-16 μg/mL. Especially, medermycin showed significant

antibacterial activity against Gram-positive bacteria, S. aureus ATCC 25923, E.

faecium ATCC 19434, B. subtilis ATCC 6633, E. faecalis ATCC 19433, with MIC

value of 0.0625, 0.0625, 0.5, and 0.5 μg/mL respectively. In case of Gram-negative

bacteria, S. entrica ATCC 14028, K. pneumoniae ATCC 10031, E. coli ATCC 25922,

and P. hauseri NBRC 3851, with MIC value of 00625, 16, 16, and 0.0625 μg/mL

respectively. Amp and Tet was positive control and DMSO was used as negative

control (data not shown). In addition, MBC show that medermycin have bactericidal

activity, not static activity (Figure S8). Medermycin showed antibacterial activity

against drug resistant S. aureus. MSSA strain CCARM 0027, CCARM 0204,

CCARM 0205, with MIC value of <0.0625 μg/mL. MRSA strain CCARM 3640,

CCARM 3089, CCARM 3090, CCARM 3634, CCARM 3635, ATCC 43300,

ATTCC 700787, and ATTCC 700788, with MIC of 0.25, 0.25, 0.25, 0.125, 0.0625,

0.125, 0.25 and 0.25 μg/mL respectively (Table 6). The Medermycin did not inhibit

the pathogenic fungi, with MIC is 64 μg/mL against C. albicans and A. fumigates, T.

rubum, and T. mentagrophytes, have >64 μg/mL. It means medermycin inhibits the

growth of bacteria selectively (Table 5). Paper disc assay showed the medermycin

have selective antimicrobial activities (Figure 6).

35

Table 5. Antimicrobial activities of compound P2 against Gram-positive bacteria, Gram- negative bacteria, and fungi

ND: No Data; c were used as positive control

No. MIC (μg/ml)

38C-P2 cAmpicillin cTetracycline cAmphotericin B

1

Gram-

positive

Staphylococcus aureus ATCC 25923 0.0625 0.125 ND ND

2 Bacillus subtilis ATCC 6633 0.0625 0.0625 ND ND

3 Enterococcus faecium ATCC 19433 0.5 0.25 ND ND

4 Enterococcus faecalis ATCC 19434 0.5 0.25 ND ND

5

Gram-

negative

Salmonella entrica ATCC 14028 0.0625 0.125 ND ND

6 Klebsiella pneumoniae ATCC 10031 16 ND 0.5 ND

7 Escherichia coli ATCC 25922 16 8 ND ND

8 Proteus hauseri NBRC 3851 0.0625 0.031 ND ND

9

Fungi

Aspergillus fumigatus HIC 6094 >64 ND ND 1

10 Trichophyton rubrum NBRC 9185 >64 ND ND 1

11 Trichophyton mentagrophytes IFM 40996 >64 ND ND 1

12 Candida albicans ATCC 10231 64 ND ND 0.5

36

Table 6. Antibacterial activities of compound P2 against MSSA and MRSA

(1-3: methicillin-sensitive Staphylococcus aureus) ND: No Data

(4-11: methicillin-resistance Staphylococcus aureus) c were used as positive control

No. Staphylococcus

aureus MIC (μg/ml)

38C-P2 cDaptomycin cVancomycin cPlatensimycin cLinezolid cCiprofloxacin 1 CCARM 0027 <0.0625 4 1 4 2 0.25 2 CCARM 0204 <0.0625 1 0.25 4 1 0.25 3 CCARM 0205 <0.0625 0.5 0.25 4 1 0.25 4 CCARM 3640 0.25 8 2 2 2 32 5 CCARM 3089 0.25 16 1 4 2 32 6 CCARM 3090 0.25 8 0.25 2 1 64 7 CCARM 3634 0.125 8 0.5 2 2 64 8 CCARM 3635 <0.0625 16 1 4 2 32 9 ATCC 3300 0.125 16 1 4 2 0.25

10 ATTCC 700787 0.25 >32 2 8 2 0.125 11 ATTCC 700788 0.25 16 2 8 2 16

37

38

Figure 6. Paper disc assay against S. aureus and C. albicans

As like MIC assay, medermycin did not show antifungal activity.

39

40

Time-kill assay

Time-kill assay were performed to examine the rate of bacterial killing by

medermycin over time. S. aureus and S. aureus MRSA 700787 were treated with 4x

(Figure S9) and 8x (Figure 7) MIC of medermycin, vancomycin, tetracycline. As

seen in Figure 7, the negative control is DMSO and show an increase in CFU counts.

Treatment with medermycin consists of a slow bactericidal phase and followed by a

regrowth with MRSA. In comparison, vancomycin had an initial rapid bactericidal

activity while tetracycline had a bacteriostatic activity with S. aureus. Vancomycin

and tetracycline had a bactericidal activity steadily with S. aureus MRSA 700787.

The means and error bars are at least three independent experiments with standard

deviations from the mean.

41

42

Figure 7. Time-kill assay

Time-dependent killing of (a) S. aureus and (b) S. aureus MRSA 700787 by medermycin and

comparator agents at 8x MIC. ●: Untreated control; ■: medermycin; ▲: vancomycin; ▼:

tetracycline

43

(a)

0 4 8 12 16 20 240

2

4

6

8

10

Time (hr)

log

10

(CF

U/m

L)

(b)

0 4 8 12 16 20 240

2

4

6

8

10

Time (hr)

log

10

(CF

U/m

L)

44

Membrane potential assay

To evaluate the effects of medermycin on S. aureus membranes, fluorescence probe

DiSC3(5) was used. When membrane is disrupted by drug, the membrane potential

is dissipated and DiSC3(5) is released, which results in an increase in fluorescence

that can be detected by fluorescence spectrometry. As shown in Figure 8, the

fluorescence probe release after nisin addition but not medermycin. Medermycin

does not alter the membrane potential, it means the target of medermycin is not

membrane.

45

46

Figure 8. Membrane potential assay

Antibiotic induced membrane depolarization and disruption, demonstrated by the

fluorescence release from S. aureus following antibiotic treatment, 4x MIC of nisin

and medermycin.

47

48

Effect of culture medium on the production of antimicrobial metabolites

Every culture solution from different medium have antibacterial activity against S.

aureus (Figure 9). The inhibition zone’s size are different, Gause’s medium is largest

and clear. Also, every culture solution have bioactivity on MIC assay but there are

no bioactivity to Gram-negative bacteria differently from medermycin (Table 7).

Also, just two of medium have bioactivity against MRSA, YPM and Gause’s,

especially Gause’s medium have good bioactivity as 1.56 μg/ml. HPLC

chromatogram show every medium made different metabolites. Compare with

medermycin peak, three of four media don’t match with medermycin but Gause’s

medium matches with medermycin. However, Gause’s medium culture solution also

don’t have antibacterial activity against Gram-negative bacteria, the peak which is

matched retention time with medermycin is not medermycin exactly (Figure S9,

Figure S10, Figure S11).

49

50

Figure 9. Agar diffusion assay against S. aureus with various media

Data shows the each inhibition zone with various medium’s culture solution

51

YPM M2 GTYB’ Gause’s

52

Table 7. MIC assay with various media

No. MIC (μg/ml)

S. aureus S. aureus MRSA 700787 K.. pneumoniae

1 YPM 3.125 12.5 >100 2 M2 50 >100 >100 3 GTYB’ 12.5 50 >100 4 Gause’s <0.39 1.56 >100 5 cAmp 0.125 ND ND 6 cVan ND 2 ND 7 cTet ND ND 0.5

ND: No data

C were used as positive control

53

Discussion

A consequence of the increase of resistant bacteria is that novel antimicrobial agents

which have activity against infectious diseases are required urgently. Marine is one

of the remarkable domain to live microorganism. One of the marine-derived

actinomycetes strain, 38C from KIOST, was showed potent antibacterial activity.

This strain was identified as Strptomyces bacillaris by 16S rRNA gene analysis. The

bioactive compound was isolated and purified to elucidate structure using various

chromatograms, MS, NMR. The compound is known as medermycin which is

known as antitumor compound. Medermycin exhibited antibacterial activity,

especially against Gram-positive bacteria. In addition, the compound showed

antibacterial activity against MRSA. The time-kill assay confirmed the ability of

medermycin, reducing the CFU slowly in 0-12 h but regrowth after 12h with MRSA.

Although compound is known, the structure is unique which is classified

pyranonaphthoquinone well known as antitumor. According to previous studies,

mechanism of medermycin as antitumor are researched well,

pyranonaphthoquinones have ability as an electron acceptor by reduction of the

quinone moiety (Buehrer and Reitemeier, 1940; Nomoto et al., 1988), but not as

antibacterial compound. I thought the target of medermycin is membrane because of

mechanism of medermycin as antitumor is alkylation and target of analog of

medermycin is membrane (Nass et al., 2017). However, membrane potential assay

identified that membrane is not target of medermycin and it needs further study to

define target of medermycin. The size of medermycin is enough small (molecular

weight: 457) and is not relevant to cell membrane, it might be permeate to inside of

54

cell and act another way of inhibiting. I think medermycin have moderate mechanism

not similar with vancomycin to bacteria because time kill assay showed CFU

decrease slowly and regrowth after 12h with 4x MIC and 8x MIC with MRSA.

According to medermycin have selective antimicrobial activity, it will be different

mechanism that apply to prokaryotes not the same mechanism as antitumor.

This is the first time to report Streptomyces bacillaris produce medermycin. The

carbon source of medium can affect secondary metabolite production. For

confirming effect of culture medium on the production of antimicrobial metabolites,

the four medium were used and confirmed bioactivity by previously described

procedure. All of the medium make bioactive compound and it seems three of four

medium don’t produce medermycin, the peak of medermycin doesn’t exist in EtOAc

extract crude on HPLC chromatogram. The Gaues’s medium looks like making

medermycin on HPLC chromatogram and MS is same with medermycin as 457, but

the antibacterial activity is quite different with medermycin. It can be analog of

medermycin or totally different compound. For identifying about this, it need to

confirm NMR spectrum.

In conclusion, this study demonstrated the potential of medermycin as an effective

therapeutic agent against bacteria, these results obtained by combinations tested in

this study. Membrane potential assay identified medermycin have another

mechanism not same with γ-actinorhodin which is the same class of medermycin

and it needs further study. Additionally, confirmed the different carbon source

effected secondary metabolites that have not same antimicrobial activity with

medermycin.

55

Supplementary Materials

Supplementary Figure 1. HPLC chromatogram of purification compound P2 ....... 58

Supplementary Figure 2. MS spectrum of compound P2 ............................................ 60

Supplementary Figure 3. 1H NMR .................................................................................... 62

Supplementary Figure 4. 13C NMR ................................................................................... 63

Supplementary Figure 5. 1H-1H COSY NMR ................................................................ 64

Supplementary Figure 6. HSQC NMR ............................................................................. 65

Supplementary Figure 7. HMBC NMR ........................................................................... 66

Supplementary Figure 8. MBC assay ............................................................................... 68

Supplementary Figure 9. 4x MIC Time-kill assay ......................................................... 70

Supplementary Figure 10. HPLC chromatogram of EtOAc extract from YPM

medium .................................................................................................................. 72

Supplementary Figure 11. HPLC chromatogram of EtOAc extract from M2

medium .................................................................................................................. 74

Supplementary Figure 12. HPLC chromatogram of EtOAc extract from GTYB’

medium .................................................................................................................. 76

56

Supplementary Figure 13. HPLC chromatogram of EtOAc extract from Gause’s

medium .................................................................................................................. 78

Supplementary Figure 14. MS spectrum of EtOAc extract from Gause’s medium80

57

58

Supplementary Figure 1. HPLC chromatogram of purification compound P2

Semi-preparative HPLC was used. (a) MeOH control (b) EtOAc extraction crude (C18

column, solvent condition ACN (+ 0.1% TFA): WATER (+ 0.1% TFA) = 25: 75, v/v, isocratic

elution, flow rate 2 mL/min, running time 40 minutes, detected by RI and UV wavelength

254, 365 nm), Semi-preparative column (Agilent ZORBAX Eclips Plus C18 4.6 X 250 mm)

59

(a)

(b)

60

Supplementary Figure 2. MS spectrum of compound P2

(a) is positive data [N+H]+ and (b) is negative data [N-H]-

61

(a)

(b)

62

Supplementary Figure 3. 1H NMR

63

Supplementary Figure 4. 13C NMR

64

Supplementary Figure 5. 1H-1H COSY NMR

65

Supplementary Figure 6. HSQC NMR

66

Supplementary Figure 7. HMBC NMR

67

68

Supplementary Figure 8. MBC assay

The X axis means medermycin concentration and Y axis means viability of S. aureus and S.

aureus MRSA 700787 at different concentration of medermycin. Bacteria alive until 2~4X

MIC but CFU decrease rapidly from 8X MIC.

69

(a)

(b)

70

Supplementary Figure 9. 4x MIC Time-kill assay

Time-dependent killing of (a) S. aureus and (b) S. aureus MRSA 700787 by medermycin and

comparator agents at 4x MIC. ●: Untreated control; ■: medermycin; ▲: vancomycin; ▼:

tetracycline

71

(a)

S. aureus 4X

0 4 8 12 16 20 240

2

4

6

8

10

Time (hr)

log

10

(CF

U/m

L)

(b)

MRSA700787 4X

0 4 8 12 16 20 240

2

4

6

8

10

Time (hr)

log

10

(CF

U/m

L)

72

Supplementary Figure 10. HPLC chromatogram of EtOAc extract from YPM

medium

(a) EtOAc extract from YPM medium only, (b) EtOAc extract with medermycin

Red arrow is peak of medermycin

73

(a)

(b)

74

Supplementary Figure 11. HPLC chromatogram of EtOAc extract from M2

medium

(a) EtOAc extract from M2 medium only, (b) EtOAc extract with medermycin

Red arrow is peak of medermycin

75

(a)

(b)

76

Supplementary Figure 82. HPLC chromatogram of EtOAc extract from GTYB’

medium

(a) EtOAc extract from GTYB’ medium only, (b) EtOAc extract with medermycin

Red arrow is peak of medermycin

77

(a)

(b)

78

Supplementary Figure 13. HPLC chromatogram of EtOAc extract from

Gause’s medium

(a) EtOAc extract from Gause’s medium only, (b) EtOAc extraction with medermycin

Red arrow is peak of medermycin

79

(a)

(b)

80

Supplementary Figure 14. MS spectrum of EtOAc extract from Gause’s

medium

(a) is positive data [N+H]+ and (b) is negative data [N-H]-

81

(a)

(b)

82

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Abstract in Korean

해양방선균 Streptomyces bacillaris 유래

항균활성물질 연구

서울대학교 대학원

농생명공학부 응용생명화학전공

배수현

미생물의 이차대사산물인 항생제는 다른 미생물의 생장을 억제하거나 방

해한다. 미생물 중 그람 양성균인 Streptomyces는 가장 주된 천연 유래

항생제 생산원이다. 최근에는 새로운 물질을 발견하기 위해서 기존의 토

양환경이 아닌 새로운 환경에서 서식하는 Streptomyces에 대한 연구가 증

가하고 있다.

본 연구에서는 한국해양과학기술원에서 제공받은 406종의 해양유래

방선균의 항미생물활성을 확인했다. 그 중, 항세균 활성을 보이는 38C

균주를 선택해 실험에 사용하였다. 16S rRNA 유전자 염기서열을 분석해

38C 균주가 Streptomyces bacillaris인 것을 확인했다. 이 균주에 대해 가

88

장 항균활성이 높게 나오는 GTYB50 배지를 사용해 28℃에서 5 일 간

배양하였다. 배양액은 유기용매로 추출 후, HPLC를 통해 항균활성물질

을 분리, 정제했다. 이 물질에 대해 질량분석법, 핵자기공명법을 이용해

구조를 규명했다. 그 결과, 물질은 항암효과가 있는 것으로 알려져 있는

pyranonaphthoquinone 계열의 medermycin으로 밝혀졌다.

정제된 물질의 항미생물활성을 측정한 결과, 그람 양성균과 음성균

에 대해 저해활성을 가지고 있으나 진균에 대해서는 저해활성을 가지

고 있지 않음을 확인하였다. 또한 저항성균주로 알려져 있는 MRSA 균

종에 대해서도 강한 활성을 보이는 것을 확인했다.

물질의 특성을 확인하기 위해 추가적인 실험으로 시간 별 박테리아

저해 곡선을 그렸다. 또한 문헌들을 참고해 메커니즘 연구를 진행했으

나 참고한 유사체들과는 다르게 medermycin은 세포막을 저해하지 않

는 것을 확인했다.

주요어: 해양방선균, Streptomyces bacillaris, 이차대사산물, 항세균

활성, 저항성 균주

학 번: 2017-23390