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1
Rapid
extraction
of
cytoplasmic
Chlorxanthomycin: A novel class
hyperthermophilic Geobacillus sp.
fluorescent
pyrene,
of Antibiotic from
Gurumurthy Dummi Mahadevan1
[email protected]
Shivayogeeswar Eshwarappa Neelagund 1†
[email protected]
Shridhar A Hucchannavar2
[email protected]
1
Department of PG studies and research in Biochemistry, Jnana Sahyadri, Kuvempu
University, Shankaraghatta, Shimoga, Karnataka, India,
2
Department of PG studies and research in chemistry, Jnana Sahyadri, Kuvempu
University, Shankaraghatta, Shimoga, Karnataka, India,
1†
Correspondence to: Dr. Shivayogeeswar Eshwarappa Neelagund. Associate Professor,
Department of PG studies and research in Biochemistry, Jnana Sahyadri, Kuvempu
University, Shankaraghatta, Shimoga, Karnataka, India Tel: 00-91-9448234456; email:
[email protected]
2
Abstract
A novel class of cytoplasmic fluorescent pigment, Chlorxanthomycin (11-Chloro- 3, 5, 7, 10tetrahydroxy-1, 1, 4, 9-tetramethyl 1-2a, 5-dihydro-1H-benzopyrene-2, 6-dione) was isolated
and characterized from hytherthermophilic alkali resistant Geobacillus sp. Iso5. Simple and
rapid method was employed in the extraction of fluorescent yellow-orange pigment. The
pigment was further characterized by Fluorescent spectroscopy, and Gas chromatography
mass spectra (GCMS). The structure was confirmed by using Infrared spectroscopy (IR) and
1
H-NMR. The isolated pigment was a pentacyclic pyrene, chlorinated molecule containing an
added methyl group with the molecular formula C 22 H19 ClO 6 and a molecular weight of m/z
426.4. The pigment was tested for antibacterial activity and Minimum inhibitory
concentration (MIC) against B.subtilis, E.coli, P. aeruginoas, S. aureus and Streptococcus sp.
revealed the selective growth inhibition on gram-positive bacteria.
Key words
Hyperthermophiles, Geobacillus sp. Iso5; Chlorxanthomycin
3
Introduction
The advancement in the investigation of hyperthermophilic bacterial groups, which
are structurally adapted at the molecular level to withstand extreme temperature, leaves us a
unique interest in various applications [1, 2]. These microorganisms produce unique
enzymes, cell components and biochemical pathways that function under conditions in which
mesophilic counterparts could not survive [3]. Because of their unusual properties, allow
them to withstand the harsh conditions typically associated with industrial processes, which
has consequently being used in several novel applications [4, 5]. Hyperthermophiles, as a
result, driven by the novel bioactive molecules found to be convenient in many applications.
Despite, certain groups of hyperthermophilic bacteria can able to produce simple to more
complex compounds in their existing environment. Especially, the production of fluorescent
pigment is predominant in member of mesophilic group unless, it is found less in
thermophiles and hyperthermophilic bacteria. Although, variety of plants, animals and
microorganisms produce fluorescent pigments. Few bacterial groups produce blue, bluegreen, yellow-orange and green-red different colored pigments, which are having different
functions and chemical properties. Instead, pigment productions on selective media are
important taxonomic tools in the differentiation and classification of bacteria [6, 7]. Most of
the industrially viable fluorescent pigments used currently are extracted from plants and other
mesophilic bacteria. It could be advantageous to produce natural fluorescent pigments from
thermophilic and hyperthermophilic bacteria due to their stability and other novel
applicability. The pentacyclic derived compounds from bacteria also exhibit fluorescent
properties, which are functionally similar to other pentacyclic pigments from plants and other
4
sources.
Chlorxanthomycin is one such pentacyclic fluorescent pigment, which is absent or less
predominant in hyperthermophiles. There are only few investigation has taken place on
widely distributed members of the genus Bacillus that produce Chlorxanthomycin [8]. These
pigments are of cytoplasmic origin and produces characteristic yellow-orange color in long
UV radiation. The Chlorxanthomycin is a pentacyclic, chlorinated, benzopyrene compound
consisting of five fused rings in a planar delocalized structure. The fluorescent pigment is
less predominant in many bacterial genera, which exhibit the characteristic yellow-orange
colored
fluorescent property.
Unlike
the
diffusible
green
fluorescent
pigments,
Chlorxanthomycin does not appear to be a siderophore. Although, it was proved too has a
selective antibiotic property on different bacterial and fungal groups.
Based on its occurrence and distribution, there is no evidence of pentacyclic derived
fluorescent pigment in thermophilic and hyperthermophilic groups especially in the members
of bacterial group 5, Geobacillus. Here we have described a simple, rapid and novel method
in
isolation
and
characterization
of
yellow-orange
fluorescent
pigment
from
hyperthermophilic Geobacillus sp. Iso5.
Material and Methods
Growth and culture condition of bacteria.
Hyperthermophilic aerobic Geobacillus sp. Iso5 was isolated from alkaline thermal
spring of Karnataka, India [9]. In brief, the bacterium was isolated in the medium containing
(w/v) 1.5% peptone, 0.2%, beef extract, 0.5% tryptone, 0.1% Nacl, 2% agar and incubated at 70
°
C with the pH value of 7.0 for 48 h. Identification of bacteria was performed previously by
conducting biochemical tests and 16S rRNA gene sequencing reveal that, 98-99% sequence
5
similarities was observed with thermophilic members of group Geobacillus [10]. The isolated
cells from the solid medium were subculture by inoculating with Luria-Bertuni (LB) broth at
°
60 C for 24 hours at pH 8.0. The mineral enrichment of LB cultured cells was then cultured in
SPYM media containing (w/v) 1 % Starch, 0.5% Peptone, 0.2% yeast extract and 0.05% Sodium
°
chloride, 1% vitamin solution and 0.5% mineral salt solution at 65 C; pH 8.0. In addition,
Tryptic Soy Agar (TSA) (HiMedia, Mumbai), Nutrient Agar (NA) (HiMedia, Mumbai) were
used for the determination of the antimicrobial properties of the pigment
Extraction of Fluorescent pigment
Simple and effective procedure was developed for the isolation of fluorescent pigment
from Geobacillus sp. Iso5. After 48 hours of incubation, the cells in SPYM medium were
harvested by centrifugation in 4 ºC at 10,000 rpm for 25 min. The cells were collected and
washed with minimum amount of five ml 50 mM phosphate buffer of pH 7.0. Approximately
°
2500 ml media yielded in 50 gm biomass was freeze dried at -45 C. Twenty five grams of
freeze dried bacterial cells were collected and washed twice with of 80% ice-cold
CH3 COCH3 and agitated for two-minute cold condition. The agitated mixture was filtered
using Whatman No 1 filter paper (Whatman, USA). The filtered mixture was kept at cold
condition and air dried. The agitated sample was extracted three times under reflux for
one hour by CHCl3 /CH3 OH (2:1) (HiMedia, Mumbai). After each subsequent reflux step,
the extracts were combined and evaporated to dryness under room temperature.
Thin Layer Chromatography
The purity of the fluorescent pigment was analyzed by thin layer chromatography
6
(TLC) using Silica Gel 120. The dried extract was dissolved in minimum amount of
CHCl3 /CH3 OH (2:1) and purity was determined under 0.5-mm silica gel sheets (Merck 60F254) using CH3 Cl-EtOAc (3:1) solvent. For the extraction from silica gel, the preparative
TLC was performed under thin (0.3 mm) Silica Gel plates (Merck, Mumbai). The fluorescent
compound was irradiated and visualized with a long-wavelength (340- 365 nm) UV lamp
(Philips, USA) [8]. The fluorescent bands was scraped off form the plate, dissolved in
minimum amount of ice-cold acetone, and filtered through Whatman No1 filter paper.
UV-visible and fluorescence spectroscopy
A UV-Visible light spectrum of the pigment was obtained with a Shimadzu UV-1800
spectrophotometer (Shimadzu, Japan). Normalized excitation of the fluorescent compound at
absorption was scanned at the range 200 to 700 nm. The emission spectra of excitation at the
fixed wavelength 400 to 600 nm was carried out with a Perkin- Elmer LS 50B luminescence
spectrometer. To assure a monochromatic light source, narrow (1.0 mm) excitation slits were
used [11, 12].
Gas Chromatography and Mass spectra (GCMS)
The GCMS profile of the fluorescent pigment was determined by the VF-5ms 30 m x
0.25mm (ID) x 0.25µm quartz capillary column with helium as the carrier gas, injector
temperature was 280°C, split ratio 15:1.0, and sample size 1.0 µL for GC condition. The
analysis was carried out by ionization mode (EI) at electron bombarding energy 70 eV, and
charging multiplier tube voltage at 500 V and the scan range was from m/z 40 to 650 at 3
scan/sec. Solvent delay at 3 min was maintained for mass determination.
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Infrared spectroscopy
The infrared spectrum was recorded on Nicolet Magna-IR 380 FTIR spectrometer
(Thermo Electron, USA) spectrophotometer using KBr pellets at 32 average scans rate.
NMR spectroscopy
1
HNMR spectra was recorded by Bruker DRX400 MHz spectrometer and acquired on a
Bruker Avance-2 model spectrophotometer using CDCl3 /DMSO as a solvent and TMS as an
internal reference [13].
Antibacterial assay
Antibacterial property of the Chlorxanthomycin pigment was determined by agar
well diffusion method. The concentration of 50 µM (1 µM= 429µg/ml) was prepared by
dissolving the appropriate weight of chlorxanthomycin in sterile distilled water. The pigment
was diluted to 50 µM concentrations and an equal amount of standard Ciproflaxin using
distilled water. The clear zone of inhibition around Bacillus subtilis (MTCC 3053),
Escherichia coli (MTCC 1698), Pseudomonas aeruginoas (MTCC 6458), staphylococcus
aureus (MTCC 6443) and streptococcus sp. (MTCC 1698) colonies indicated the
antibacterial property of the pigment. For MIC, the dried pigment was diluted in sterile
distilled water to obtain the final concentration of 50 µM to 100 µM. To test minimum
inhibitory concentration, a small amount of a bacterial suspension in 10 mM phosphate
buffer (pH 7.0) was spread over nutrient agar pH 7.0. The final concentration of pigment was
filled into the wells Petri plate. The growth was monitored for 48 hours and the zone of
inhibition was measured around the wells.
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Results
Extraction and characterization of the Pigment
Simple procedure for isolation of water insoluble fluorescent pigment from
Geobacillus sp. Iso5 was determined to produce Yellow-orange color under long wavelength
of UV intensity (Figure. 1). The CHCl3 /CH3 OH extraction (2:1) of pigment under reflux
condition yield bright yellow-orange colored spot on silica gel plate. The purity of the
extraction protocol was analyzed by preparative thin layer chromatography also revealed the
presence of single yellow-orange colored spot (Rf: 5.3 cm) on the thin silica gel plates. The
pigments fluorescent property was determined by dissolving in acetone, dimethyl sulfoxide,
methanol and pyridine also revealed the presence of same yellow orange fluorescent color.
The molecular mass of the pigment was analyzed by using GCMS revealed the
presence of one major peak at m/z 426.4 (Figure. 2). However, based on previous literature
+
and chemical formula (C 22 H19ClO 6 +H ) the molecular mass was considered to be 409.
The fluorescent spectroscopic measurement of the yellow-orange colored pigment absorption
spectrum from 200-700 nm had six major peaks at 260, 361, 392, 421, 485 and 521 nm
(Figure. 3a). Except peak 421 nm, all other major peaks did not showed any florescence
emission. Further excitation at 400 to 600 nm, the emission wavelength was shown two
excitation peak was observed at 421 nm and 476 nm (Figure. 3b). This concluded that,
Chlorxanthomycin of Geobacillus sp. Iso5 is a conjugated pigment.
1
The H NMR (DMSO-d6) spectrum of Chlorxanthomycin showed the following
characteristics: δ ppm = 0.9 (s, 32H, CH3), 2.7 (s, 3H, CH3), 3.5 (s, 6H, gem dimethyl), 4.8
9
1
(s, 3H, 4H, - OH) (Supplement figure). Interpretation of the H NMR spectrum indicated the
presence of an isolated methyl group, a gem dimethyl, -OH groups and one isolated aromatic
protons, each occurring as a singlet. The IR spectrum of chlorxanthomycin showed
-1
absorbance at IR (V max, cm KBr) = 786 (C-Cl stretching), 1640 (C=C), 1780(C=O), 2080
(Ar C-H stretching), 3460(-OH stretching vibration) (figure 4). Absorption clearly indicates
that the molecule is aromatic. Although this compound has carbonyl groups, it does not have
-1
the IR frequency around 1,780 cm that is typical for stretching of a carbonyl group. Thus,
the carbonyl groups must be present in the form of enols or hydroxy, unsaturated ketones
1
(since the result of H NMR ruled out aldehyde, carboxylic acid, and ester), and the carbonyl
and hydroxy groups are intramolecularly hydrogen bonded. The IR absorption and
fluorescence suggest that chlorxanthomycin may have a large conjugated aromatic system,
such as a pentacyclic phenalenone structure.
Based on these spectral studies, it was determined that the molecule is a benzopyrene
compound consisting of five fused rings in a planar delocalized structure. There are four
hydroxyl groups, two carbonyls, four methyl group, a gem dimethyl groups, and chlorine
bonded around the edge of the fused ring system. The deviations of the core carbon atoms
from the least squares plane are consistent with a small saddle deformation. The groups
bonded around the core followed the same pattern of deviation from planarity as the atoms to
which they were bonded (Figure. 5).
The Chlorxanthomycin was shown antibacterial property against gram positive Bacilli
at the concentrations of 50 mM. B. subtilis was inhibited by showing 4.6±0.1 mm zone of
inhibition around the well. The detailed antibacterial activity of Chlorxanthomycin was
10
presented in Table 1. However, MIC values indicate the lower concentration of
Chlorxanthomycin also proves inhibitory to B. subtilis at 50 to 60 µM concentration. On the
other hand, Escherichia coli, Pseudomonas aeruginoas, staphylococcus aureus and
streptococci were also inhibited by Chlorxanthomycin pigment at the concentration ranging
from 60 to 100 µM. The detailed inhibitory activity of Chlorxanthomycin on different grampositive bacteria is described in Table 2.
Discussion
Extremophiles, isolated from the extreme ecological niches, are well adapted to
unfavorable environmental factors and have huge biotechnological potential [4, 14, 15].
Especially, the biologically active substances are synthesized by extremophiles, currently
sought to be important for their ability to survive and proliferate. These compounds are
generally nonessential for growth of the organism and are synthesized with the aid of genes
involved in intermediary metabolism. Among bioactive compounds of microbial origin have
been characterized, limited number of compounds is used for biomedical, agricultural and
industrial applications. One such natural bioactive compound is fluorescent pigment with
potent antibiotic properties.
The production of fluorescent pigments by groups, especially abundant with fluorescent
Pseudomonads was reported [7, 16]. These present adjacent to the laimosphere,
spermosphere and rhizosphere in the soil. [17-20]. The Pseudomonas fluorescens produces
yellow-green fluorescent pigment (YGFP) from brewery waste yeast with and without
mineral salts in the medium [21]. Some authors described the properties of water soluble
fluorescent pigment of the Pseudomonads called Pyoverdine [17, 22]. There are only few
reports available on the fluorescent pigment production from Bacillus sp. [23, 24]. Although,
the influence of some natural and synthetic medium for the production of blue, blue- green,
11
yellow-orange and green-red fluorescent colored pigments from B. fluorescens spp., B.
viridians and B. fluorescens liquefaciens [7, 16, 18, 25]. Further, a previous study also
showed that ordinary nutrient media for B. pyocyaneus, B. fluorescens liquefaciens, and B.
fluorescens non-liquefaciens accompanied by the diffusion of a green or greenish blue
fluorescence throughout the culture [26] However, there are only few Bacillus spp. produces
yellow green diffusible pigments were reported.
The first report on a Chlorxanthomycin produced by a member of the genus Bacillus has
gained the major attention to words bacterial fluorescent pigments [8]. It has a conjugated
ring system with strong intermolecular hydrogen bonds between the hydroxyl and carbonyl
groups. Except for gem dimethyl groups at C-1, the polycyclic aromatic ring system belongs
to the benzo [cd] pyrene structural class and is responsible for the strong fluorescence
activity. It was also reported that, Chlorxanthomycin exhibit good antimicrobial properties
against many bacteria, fungal groups [8]. The Chlorxanthomycin is likely to that
resistomycin, resistoflavin, itamycin pigments which exhibit similar structure. However,
Resistomycin is a fluorescent pentacyclic, highly conjugated pigment and having potent
antibacterial, antiviral and anticancer properties [27, 28].
There is no evidence of pentacyclic, chlorinated fluorescent pigments from thermophiles
and hyperthermophiles. These derivatives have structural similarities with Chlorxanthomycin
of mesophilic Bacillus sp. isolated from agriculture field [8]. Like Chlorxanthomycin of
Bacillus sp., it produces yellow-orange fluorescent intensity and has selective antibacterial
properties. However, the difference in the molecular weight was attributed by the addition of
an extra methyl group at C-4 of the pentacyclic ring system
Conclusions
12
This is the first report of a fluorescent, chlorinated, pentacyclic pyrene, natural compound
produced by a member hyperthermophiles. We have employed simple and easy procedure for
the extraction of the fluorescent pigment from Geobacillus sp. Iso5. Chlorxanthomycin from
Geobacillus sp. was structurally and functionally similar to the mesophilic Bacillus sp. It also
has a good antibacterial property on many gram positive bacteria. Due to lack of description
on pentacyclic-derived pigments from hyperthermophiles, we believe that it has wider
industrial and therapeutic applications.
Acknowledgme nt
The authors would like to thank all the research staffs and members of Department of
Biochemistry, Kuvempu University. Also, like to thank Mr. Rohith Kumar H.G, Research
Scholar, Department of PG studies and Research in Biochemistry, Davangere University for
providing the fluorescence spectroscopic facility.
Conflict of interest
No conflict of interest declared
.
13
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15
Table 1: Antibacterial activity of Chlorxanthomycin
Concentration
(50µM)
Chloroxanthomycin
Ciprofloxacin
B. subtilis
(MTCC 3053)
4.6±0.1
08±0.1
Organisms
E. coli
P. aeruginoas sp
(MTCC 1698)
(MTCC 6458)
3.9±0.1
09±0.1
3.2±0.1
09±0.1
S. aureus
(MTCC 6443)
Streptococcus
(MTCC 1698)
3.8±0.1
11±0.1
1.8±0.1
7.5±0.1
*The values of each constituents’ consisted of Mean ± SE of three replicates. The concentration of 50µM (1 µM= 426µg/ml) was prepared by dissolving the appropriate weight of chlorxanthomycin in
sterile distilled water.
16
Table 2: Minimum inhibitory concentration (MIC) of Chlorxanthomycin
Pigment
Concentration
(µM)
50
60
70
80
90
100
Organisms
B. subtilis
(MTCC
3053)
4.5±0.1
4.7±0.1
5.1±0.1
5.3±0.1
5.3±0.1
5.3±0.1
E. coli
(MTCC 1698)
P. aeruginoas
(MTCC 6458)
S. aureus
(MTCC 6443)
Streptococcus sp
(MTCC 1698)
4.0±0.1
4.1±0.1
4.3±0.1
4.4±0.1
4.5±0.1
4.7±0.1
3.8±0.1
4.0±0.1
4.1±0.1
4.2±0.1
4.5±0.1
4.7±0.1
3.8±0.1
4.0±0.1
4.5±0.1
4.5±0.1
4.7±0.1
4.9±0.1
2.1±0.1
3.1±0.1
3.6±0.1
3.9±0.1
4.0±0.1
4.4±0.1
Ci profloxacin
(100 µM)
12±0.1
13±0.1
13±0.1
16±0.1
13±0.1
Sterile Distilled
Water
(Control)
00±0.1
00±0.1
00±0.1
00±0.1
00±0.1
*The values of each constituents consisted of Mean ± SE of three replicates. The concentration of µM (1 µM= 426µg/ml) was prepared by dissolving the appropriate weight of chlorxanthomycin in
sterile distilled water.
17
FIGURES
Figure 1. Fluorescent property of extracted Chloroxanthomycin from thermophilic Geobacillus
sp. Iso5
A
B
*Chloroform/methanol extracts “B” and control solvent “A”. Dried extract was suspended in chloroform/methanol (1:1), and visualized by longwavelength UV intensity (350 nm). “A” indicates the control system containing only suspension solvent without extract did not shown any
fluorescent property.
18
Figure 2 : Gas-chromatography and mass spectra of the purified Chloroxanthomycine showing
an intense peak at 426.4 with 90% average intensity
19
Figure 3 (A & B): Absorption, excitation, and emission spectra of the fluorescent compound
600
400
521.04 , 211.346
421.07 , 606.136
485.00 , 207.355
Intensity (a.u.)
800
392.03 , 638.280
295.93 , 987.159
361.07 , 713.461
1000
279.06 , 957.704
(A)
260.95 , 661.226
247.03 , 915.071
dissolved in ethanol
200
0
200
300
400
500
Wavelength (nm)
600
700
(B)
Intensity (a.u.)
800
600
400
475.97 , 127.774
421.06 , 368.155
1000
200
0
200
300
400
500
Wavelength (nm)
600
700
*(A) Normalized excitation of the fluorescent compound at absorption range 200 to 700 nm. (B) The emission spectra at excitation 400 to 600
exhibit two major peaks at 421 nm and 475 nm respectively
20
Figure 4: Infrared spectrum of the Chloroxanthomycine from Geobacillus sp. Iso5
0.0682
0.065
0.060
0.055
0.050
0.045
0.040
0.035
A
0.030
0.025
0.020
0.015
0.010
0.005
0.000
-0.0019
4500.0
4000
3600
3200
2800
2400
2000
cm-1
1800
1600
1400
1200
1000
800
650.0
21
Figure 5: Predicated structure of Chlorxanthomycin from Geobacillus sp. Iso5 with chemical
nomenclature of 11-Chloro- 3, 5, 7, 10-tetrahydroxy-1, 1, 4, 9-tetramethyl 1-2a, 5-dihydro-1Hbenzopyrene-2, 6-dione
The 1H-NMR spectra revealed the presence of an isolated methyl group, a gem dimethyl, -OH groups and one isolated aromatic protons, each
occurring as a singlet. And the estimated molecular mass was m/z = 429.14 (M+)