REVIUW JURNAL

Isolation of Aromatic Polyketide Producing Soil

Streptomyces Using Combinatorial Screening Strategies

Sunita Bundale1*, Deovrat Begde1 , Nandita Nashikkar1 , Tukaram Kadam2 , Avinash

Upadhyay1

OLEH :

IDAWATI

917312906201.003

FAKULTAS SAINS DAN TEKNOLOGI

S1 FARMASI

INSTITUT TEKNOLOGI DAN KESEHTAN AVICENNA

KENDARI

2020
 ABSTRAK

Streptomyces, the soil dwelling filamentous genera of actinomycetes, are known to

produce aromatic polyketides, which exhibit antibacterial, antifungal, antiviral and anticancer
activities. The purpose of the current study was to screen soil samples for aromatic polyketide
producing Streptomyces spp. using a judicious combination of conventional and molecular
screening methods. A total of 54 isolates were isolated from soil samples collected from in and
around Nagpur, Maharashtra, India. These isolates were characterized by conventional
methods. The bioactivity of the isolates was assessed by agar cross streak method against
B.subtilis, M.luteus, E.coli, P. aeruginosa, C.albicans and A.niger. Out of the 54 isolates,
31isolates (57%) showed activity against one or other test organism. These 31 bioactive
isolates were subjected to secondary screening using agar well diffusion method, whereby 21
isolates were consistent in their bioactivity. The production of aromatic polyketides by these
21 bioactive isolates was determined by spectroscopic studies and chemical screening of
their organic extracts. 11 isolates, which tested positive, were tested for PCR amplification
of KSα fragment of type II PKS gene using degenerate primer pair act04/act06.The obtained
results showed that 9 isolates (16.66%) showed the ability to produce aromatic polyketides.
Out of these 9 isolates, five potent isolates were identified using 16S rRNA gene sequencing
and their accession numbers have been deposited in GenBank. The identity of these isolates
confirmed their ability to produce aromatic polyketides. This study indicated that
combinatorial screening strategies can be used for correct identification of aromatic polyketide
producing culturable Streptomyces spp from soil.
 Introduction

Soil actinomycetes, especially those that belong to the genus Streptomyces, have been
the focus of intensive research. for the past several decades. The interest in Streptomyces arose
from the finding that this group of bacteria seems to have the ability to produce a large variety
of different bioactive compounds that have a wide spectrum of activity (Metsä-Keteläet al.
2002). Polyketides belong to a group of such bioactive compounds, of which Streptomyces is
a well known producer. Most of the polyketides have been isolated from Streptomyces
species indicating that they have a tremendous potential to produce numerous different
polyketides (Liu et al. 2012). Aromatic polyketides, an important member of the family of
polyketides and synthesized by type II polyketide synthase (PKS), exhibit a wide array of
biological activities including antibacterial, antitumor, antiviral and enzyme inhibitory
activities (Sun et al. 2012), and afford some of the most common antibiotics and
anticancer drugs currently in clinical use, e.g. tetracyclines and anthracyclines.

Streptomycetes are among the most numerous and ubiquitous soil bacteria (Hodgson
2000; Bentley et al. 2002). Many screening programs have been carried out leading to the
discovery of numerous new bioactive molecules, and these molecules subsequently have
found their way into various clinical uses ranging from control of infections to cancer
treatment (Metsä-Keteläet al. 2002). Bioactivity guided screening is the oldest and most
common screening strategy (Monaghan and Tkacz 1990; Liu et al. 2011). But, at times, many
novel compounds, which may be activ against other targets, are overlooked. To overcome this
problem, many alternative screening strategies are tried out. One method involves the use of
spectroscopic techniques for the analysis of crude extracts and prediction of bioactive
compounds. Characterization of Streptomyces isolates and their extracts using UV-visible
spectra has been carried out by many researchers (Thakur et al. 2007; Saadoun et al. 2009;
Fguira et al. 2012). Another simple and inexpensive method for visualizing the secondary
metabolite pattern of a strain is the chemical screening approach given by Taddei et al.
(Taddei et al. 2006). Using this method, various new secondary metabolites have already been
discovered (Zähner et al. 1988; Taddei et al. 2006), and reliable, rapid and efficient
protocols for screening of extracts of microorganisms have been established over the years
(Iwai and Omura 1982; Magome et al. 1996; Fuchser and Zeeck 1997).

Aromatic polyketides, the main focus of this study have gained importance for
their antibacterial (e.g. oxytetracycline), antifungal (e.g. pradimicin), antiviral (e.g. A-74528),
antiparasitic (e.g. frenolicin) and anticancer (e.g. doxorubicin) activities (Das and Khosla
2009; Zhan 2009) and are synthesized by the enzyme type II polyketide synthase (PKS).
 Type II PKS consists of three or more enzymes that act in an iterative manner (Katz
andDonadio 1993). The core module in all type II PKS gene clusters is composed of
ketoacyl-synthase (KSα), chain length factor (KSβ) and acyl carrier protein (ACP) (Staunton
and Wilkinson 2001). Moreover, cyclase is responsible for the cyclization of aromatic
polyketides (Komaki and Harayama 2006). Thus, KSα and cyclase gene can be used as
markers for the screening of type II polyketide compounds (Sun et al. 2012). Metsa-Ketela et
al. used a method based on PCR amplification of KSα fragment of approximately 450bp
corresponding to the minimal PKS cluster (Metsä-Ketelä et al. 1999). The detection and
sequencing of PKS genes has since then often been used as a screening method for the
discovery of novel polyketides (Ayuso et al. 2005; McAlpine et al. 2005; Banskota et al.
2006). Screening of Streptomyces cultures has for decades yielded novel industrially important
products. However, the rate of discovery has not kept pace with resource expenditure and
advance in technology (Strohl 2000). Better combinatorial methods are clearly needed for
more efficient identification and screening of soil Streptomyces cultures (Wawrik et al.
2005).

 Materials and Methods

1. Collection of soil samples

The soil samples were collected from various locations in and around Nagpur,
Maharashtra, India, from up to a depth of 20 cm after removing approximately 3 cm of
the surface soil. These samples were subjected to heat treatment at 70ºC for different
time intervals ranging from 15 min to 1 hour in a hot air oven. Each soil sample (1 g) was
added to a conical flask containing 100 ml of sterile water and few drops of Tween 80. The
flasks were shake for 30 minutes in an orbital shaker incubator at 28ºC and used for
isolation.

2. Test organisms

The target strains used for screening antimicrobial activity were procured from
microbial type culture collection and gene bank (IMTECH, Chandigarh, India) and were:
Micrococcus luteus MTCC 106, Bacillus cereus MTCC 430 Escherichia coli MTCC 443,
Pseudomonas aeruginosa MTCC 741, Candida albicans MTCC 227, and Aspergillus niger
MTCC 282.

3. Chemicals and Media

All chemicals and solvents were of analytical grade and purchased from Merck,
Germany and Culture media from Hi-media, Mumbai, India. Standard doxorubicin was
obtained from Sigma-Aldrich (Cat # 25316-40-9)
4. Isolation, characterization and storage of the isolates (Streptomyces spp.)

Each stock culture of soil sample was allowed to settle and serial dilutions up to

10 -4 were prepared using sterile physiological saline and vortexed at maximum speed. An
aliquo of 0.1 ml of each dilution was taken and spr ead evenly over the surface of
Actinomycetes isolation agar, ISP 2 media and Starch-Casein agar medium. The three
media were supplemented with Amphotericin B (50 µg/ml) to inhibit fungal contamination.
Plates were incubated at 28°C, and monitored after 48, 72, and 96 hours. Plated dilutions
that gave 15-150 colonies were chosen for further isolation. Repeated streaking on ISP 2
medium (Hi-Media, Mumbai, India) plates led to isolated bacterial colonies that showed a
Streptomyces like appearance. Streptomyces colonies were recognized on the basis of
morphological characteristics by light microscopy. (Olympus, GX10).

These colonies were cultured by streak plate technique on Glycerol Yeast extract
agar, Glycerol-Asparagine agar (ISP 5), Yeast Malt extract agar (ISP 2) or Starch-Casein
agar plates as previously described (Shirling and Gottlieb 1966; Waksman 1967) and
incubated at 27-30°C for 1-2 weeks before use. Selected Streptomycete colonies were
transferred from the plates onto respective agar slants and incubated at 28°C for 14 days.
Slants containing pure cultures were stored at 4.0°C until further examinations.

Streptomyces colonies were characterized morphologically and physiologically

following the methods given in the International Streptomyces project (ISP) (Shirling and
Gottlieb 1966). The isolates were identified as species belonging to the genus
Streptomyces by analyzing their morphological characteristics (Locci 1989) and by
biochemical tests (Kutzner et al. 1992). The isolates were identified morphologically
to the genus level by comparing the morphology of spore bearing hyphae with entire
spore chain as described in Bergey’s Manual (Locci 1989). Lysozyme sensitivity was
determined by inoculating the isolates into glycerol broth with and without lysozyme
(50 μg/ml). Flasks were incubated at 28°C in a shaker incubator and examined daily for
growth up to 7 days (Berd 1973; Kiska et al. 2002). Presence of LL-DAP in the cell
walls of the isolates was assessed by the method devised by Lechevalier (Lechevalier and
Lechevalier 1970).

5. Preliminary Screening by Agar Cross Streak method

 The Streptomyces isolates were assessed for their capability of producing bioactive
compounds by agar cross streak method (Peela et al. 2005). Each of the isolate was streaked as
a dense straight line on nutrient agar plate incubated at 27°C for 6 days (144 h). After the 6th
day, test bacterial strains were streaked at right angle, but not touching each other, and then
incubated at 37°C for 24 h. For test fungal strains a PDA (Potato Dextrose Agar) plate was
used and incubated at 28°C for 48 h. The Streptomyces isolates producing bioactive
compounds inhibited the growth of test microorganisms and were selected for further
experiments (Dhanasekaran et al. 2009). The zone of inhibition against each test organism
was noted (Haque et al. 1996).

6. Secondary screening by agar well diffusion method

Isolates that showed activity against test organisms in the preliminary screening
by cross streak method wer selected for secondary screening. These isolates were grown
in submerged culture in 250 ml flasks containing 100 ml of PDB (Potato Dextrose Broth)
medium (Pundir and Jain 2006). The resulting culture broths obtained following growth of
each isolate in the culture media were sep arated from the mycelium by centrifugation at
6,000 rpm (REMI C-24 Plus) for 15 min. The supernatant, sterilized by filtration and
vacuum concentrated five-fold in Vacufuge Plus (Eppendorf, North America) was used for
estimation of antimicrobial activity by agar well diffusion method (Grammer 1976; Rojas et
al. 2006; Wiegand et al. 2008). 50 µl of the supernatant of each isolate was used for
determining the antibacterial activity. The diameters of zones of inhibition were determined
after 24 h of incubation at 37°C for bacteria and 28°C for yeasts. Each experiment was
repeated three times and mean value of the diameters of the zones of inhibition was
calculated. Negative controls contained sterile liquid culture medium.

7. Organic extraction of
bioactivecompounds

Crude antimicrobial compound was recovered from culture filtrate of each

bioactive isolate by solvent extraction with ethyl acetate, 1:1 (v/v). The dried crude
extracts were resuspended in methanol and used for physico -chemical screening and also
for antimicrobial testing using agar well diffusion method. 15 µl of 1 mg/ml dried
extract was used for this purpose and the diameters of zones of inhibition obtained were
measured.

8. Physico-
chemicalscreening

The absorption spectrum of bioactive crude organic extracts in methanol were

recorded in the UV -visible region (190-1100 nm) by using a biospectrophotometer
 (Model BL 198, Elico, India) and observed for peaks in 230 -280 nm range and 400-500
nm range (Fiedler 1993). Doxorubicin, an anthracycline was used as a standard. Chemical
screening of the crude extracts was carried out by the method described by Taddei (Taddei
et al. 2006). TLC screening was performed on readymade silica gel plates (Merck,
Germany, Cat # 1.05554.0007). Each TLC plate (20 × 20 cm) was spotted with samples
of the various microbial crude extracts prepared as described above. Chromatograms were
developed using CHCl3: MeOH: aqNH3 (85:14:1), or Benzene: Acetone: methanol
(100:10:1), methanol: chloroform: acetic acid: water (80:20:2:0.2) Chloroform: Heptane:
methanol (10:10:3) as the solvent systems.

Detection of secondary metabolite patterns was performed using a UV lamp at 254

and 365 nm, as well as by direct staining using coloring reagents. The staining reagents
used were formaldehyde-H2SO4 reagent and naphthoresorcinol reagent (Merck 1970;
Taddei et al. 2006).

9. MolecularScreeni
ng

Total genomic DNA from each bioactive Streptomyces strain was isolated by the
standard method of Hopwood (Hopwood et al. 1985) and purified using DNA purification
kit (Bangalore Genei, Merck).

Purified genomic DNA was used as a PCR template. The nucleotide sequences of
the degenerate primer set used for the amplification of KSα genes were 5'-GAT GGT
CTC CAC CGG CTG C-3' and 5'-GTC TCG TGG CGG TCG TTC TGC -3'. The
primers designed were used to amplify a 0.47 kb product corresponding to an internal
fragment from within the KSα gene. The design strategy employed embedding a 19 base
pair forward primer (act04f) in the active site of the KSα gene at nucleic acid position 595-
614 (numbers correspond to Streptomyces coelicolor GenBank accession number:
X63449), the corresponding 21 base pair reverse primer (act06r) was positioned at 1048-
1069 flanking the KSα gene. The reaction mixture contained 0.2 mM of each dNTP, 0.5
% (v/v) DMSO, 1 mM of degenerate primers, 12.5 ng of genomic DNA and 0.6 units of
Ex-Taq Hot Start Version in a final volume of 25 µl of 1×Ex-Taq PCR buffer. PCR
reaction was started with denaturation at 96ºC for 5 min followed by a total of 25 cycles
that consisted of denaturation at 96ºC for 30 sec, annealing at 58ºC for 30 sec and
extension at 72ºC for 1 min. The reaction was terminated with a final extension at 72ºC
for 5 min. The PCR reaction was performed on a thermal cyclers (Bio-Rad Laboratories,
California, USA). The size of PCR products was around 470 bp in length.
 10. Identification of potential Streptomyces isolates

The genomic DNA of the potential Streptomyces isolates was obtained by following the
method proposed by David Hopwood (Hopwood et al. 1985). The 16S rRNA gene was
amplified with primers 8-27f (5'- AGAGTTTGATCCTGGCTCAG-3') and 1500r
(5'AGAAAGGAGGTGATCCAGCCA-3'). The amplified DNA fragment was separated on 1
% agarose gel, eluted from the gel and purified using QIA quick gel extraction kit (Qiagen).
The purified PCR product was sequenced with 27f, 519r (5-GWATTACCGCGGCKGCTG-
3), 1087r (5-CTCGTTGCGGGACTTAACCC-3), 530f (5-TTCGTGCCAGCAGCCGCGG-3),
945f (5- GGGCCCGCACAAGCGGTGG-3) and 1492r respectively (Escherichia coli
numbering system). The rDNA sequence was determined by the dideoxy chain-termination
method using the Big-Dye terminator kit using ABI 310 Genetic Analyzer (Applied
Biosystems, USA). Almost complete sequences were determined and compared with those of
other closely related taxa retrieved from the GenBank database. Phylogenetic trees were
constructed by NJ plot (Perrière and Gouy 1996). A sequence similarity search was done
using GenBank BLASTN (Altschul et al. 1997). Sequences of closely related taxa were
retrieved; aligned using Clustal X programme (Thompson et al. 1997) and the alignment was
manually corrected. For the neighbour-joining analysis (Saitou and Nei 1987), the distances
between the sequences were calculated using Kimura's two-parameter model (Kimura 1980)
Bootstrap analysis was performed to assess the confidence limits of the branching (Felsenstein
1985). The 16S rRNA gene sequences have been deposited in the Genbank and accession
numbers have been acquired.

 R 1. Isolation and caracterization

e
s of isolates
u
l
s
t Using the selective media and cultivation conditions described earlier,
a total of fifty four different putative Streptomyces isolates were recovered
from the soil samples. All isolates were slow growing, aerobic with wrinkled,
folded colonies typical of Streptomyces genus (Locci 1989). The colonies
exhibited aerial and substrate mycelium of different colors and possessed an
earthy odour. On the basis of the color of the substrate mycelium and aerial spore
mass, the isolated strains were categorized into six color series and named
accordingly.
 (<1%) color series. The reverse side color and soluble pigment were examined on
he ISP 2 media plates as recommended by Shirling and Gottlieb (Shirling and
iso Gottlieb 1966). Out of 54 Streptomyces isolates, 36 (66.66 %) strains showed
s distinctive reverse side pigment, and 26 (48%) strains produced soluble pigments.
bel Melanin production was examined on ISP 6 and ISP 7 media plates. The
ing to formation of melanin was indicated by the appearance of a dark blue to black
gra colour in the agar medium after incubation on peptone- yeast extract agar (ISP 6)
bro and tyrosine agar (ISP 7) for 7 days. 18 isolates (33.33%) were found to produce
an melanin. The cultural characteristics (Pigment production) and morphological
ye characteristics of the different Streptomyces isolates are presented in Table 1.
co
ser All the Streptomyces isolates were acid-fast negative and Gram-stain
we positive. In microscopic analysis all the isolates exhibited typical filamentous
eq structures. All of these isolates fitted the genus description as reported by several
y
investigators (Shirling and Gottlieb 1966; Williams et al. 1983). The fact that they
abu
nt belong to the Genus Streptomyces was confirmed on the basis of their sensitivity
(22 to lysozyme. Lysozyme sensitivity was defined as poor or no growth in the
foll
presence of lysozyme as compared to that of glycerol control flasks. The presence
ed
wh of LL -DAP in the cell walls of the isolates further confirmed that the isolates
(16 belonged to the Genus Streptomyces. DAP spots on the chromatogram were olive
),
green which faded to yellow. The L- isomer moved several centimeters ahead of
(11
) a the meso-isomer.
vio
Table 1: Morphological and cultural characteristics of the Streptomyces isolates

Color series
Gra Brown Yellow White Re Violet Total
No. of isolates 12 12 12 9 6 3 54

Pigment

Reverse side 8 9 11 - 6 2 36(66.6

Soluble pigment 4 8 8 1 4 1 26(48%
Starch Hydrolysis 4 2 3 2 3 - 14(26%
Numbers in parentheses represent the percentage out of the total isolates

2. Screening for bioactivity

When the 54 Streptomyces isolates were subjected to primary screening using agar
cross streak method, 31 isolates showed antimicrobial activity against one or other test
organism. 14 isolates showed only antibacterial activity, 2 isolates showed only antifungal
activity and the remaining 15 isolates showed both antibacterial and antifungal activity.
Except isolates B11, W2, W7 and G10 all antibacterial isolates showed activity only against
Gram-positive bacteria. The most bioactive isolates belonged to red and violet series (100%)
followed by gray (53%), yellow and white (50% each) and brown and cream (37.5%)
showing least bioactivity. Only antifungal activity was exhibited by two isolates namely
isolate B1 and isolate B12, both belonging to Brown series. Amongst the isolates which were
broad spectrum, antibacterial activity was more predominant as compared to antifungal
activity except isolate Y9, which had more potent antifungal activity than antibacterial
activity. Four isolates namely, isolate V3, G6, B8 and Y7 did not exhibit any anti Candida
activity, though they were active against filamentous fungi, such as Aspergillus niger. Figure
1 and Figure 2 show the antibacterial and antifungal activity respectively of some isolates as
obtained by cross streak plate method.

All the 31 Streptomyces isolates selected in primary screening were subjected to

secondary screening against B. cereus, E.coli and C. albicans. Only 21 isolates were found to
be positive with isolate R1 and Y8 being the most potent isolates showing a zone of
inhibition of 21 mm and 27mm respectively. Table 2 shows the diameters of zones of
inhibition of the bioactive isolates as obtained by secondary screening. The remaining 10
isolates (32%) failed to show bioactivity in liquid medium. These 21 isolates were selected for
physico chemical screening to detect their ability to produce aromatic polyketides.

Table 2: Secondary screening by agar well diffusion method

Isolate No. B.cereus C.albicans

R1 21 ±0.56 18 ±0.34
B1 - 10 ±0.09
V1 7 ±0.42 -
G11 18 ±0.34 -
V3 3 ±0.44 -
B11 14 ±0.19 2 ±0.12
Y1 5 ±0.09 5 ±0.43
W1 - -
W2 - -
R2 18 ±0.53 6 ±0.25
B12 - 15 ±0.60
G3 17 ±0.22 -
R3 14 ±0.15 -
G4 - -
Y3 - -
Y9 2 ±0.06 14 ±0.37
Y10 19 ±0.20 -
B5 - -
W6 4 ±0.60 -
W7 - -
B9 - -
G7 18 ±0.17 -
B7 - -
B8 8 ±0.13 -
R4 18 ±0.41 5 ±0.09
G10 9 ±0.08 15 ±0.50
Y7 - -
Y12 - -
R5 7 ±0.09 -
Y8 27 ±0.14 20 ±0.41
R6 16 ±0.56 -
 Table 3: Physico chemical screening of bioactive Streptomyces isolates

Isolate No. λ max Formaldehyde- H2SO4 reagent Naphtho-resorcinol reagent

R1 277, 307, 532 + +

B1 287, 417, 440 + +
V2 282, 307, 532 + +
G11 230, 306, 581 + +
V3 297, 332 + -
B11 260, 316 + -
Y1 230, 372 + -
R2 324 - -
B12 287, 412, 447 + +
G3 294,331 - -
R3 257, 527, 553 + +
Y9 217, 297 - -
Y10 227, 342 + -
W6 317 - -
G7 247, 267, 412, 437(sh) + +
B8 242, 283, 537 + +
R4 342, 397 - --
G10 302 - -
R5 288, 325, 352, 417, 502, 587 + +
Y8 282, 312, 422, 442 + +
R6 283, 493, 525 + +
3. Physicochemical screening

Out of the 21 bioactive Streptomyces isolates selected for physicochemical

screening, the extracts of 19 isolates showed UV absorption maxima in the range
208 and 288nm indicating the presence of an aromatic nucleus. When the extracts
of these 19 isolates were chemically screened, 15 showed a color reaction with
formaldehyde-sulphuric acid reagent confirming the presence of an aromatic
nucleus in their structure. The UV- Visible spectra of at least 12 extracts showed an
additional peak in the 400-500nm range, which could indicate the presence of an
extended chromophore as seen in aromatic polyketides (Doyle et al. 1979).
Chemical screening using naphthoresorcinol reagent showed the presence of sugars
in the extracts of 11 isolates (Table 3).

4. Moleculer screening

11 isolates (R1, B1, V2, G11, B12, R3, G7, B8, R5, Y8 and R6) which were
demonstrated to be potential aromatic polyketide producers on the basis of
physico-chemical screening were further subjected to screening for KSα fragment
of type II PKS gene using PCR. Under the optimized PCR conditions, a single band
about 470 bp size was amplified from S. nogalater using the primer pair
act04/act06. This was considered to be the standard. Under the same conditions,
except isolates G11 and B8, all other 9 Streptomyces isolates were found to be
positive in PCR showing an amplicon of 470 bp confirming the presence of KSα
fragment of PKS type II gene in their genomes as seen in Figure 3. Figure 4 is a
schematic representation showing screening of aromatic polyketide producing
Streptomyces species.
Figure 3. PCR amplification of KSα fragment of the PKS gene cluster. PCR
amplification of 0.47 kb KSα internal fragment of the PKS gene cluster from
Streptomyces soil isolates. (A) Lane 1: 500bp Molecular weight marker, Lane 2:
isolateB8, Lane 3: isolateB1, lane 4: isolate V2, lane 5: isolate G7, lane 6: isolate
B12, lane 7: isolate R3, lane 8: isolate R1 (B) Lane 1: 500bp Molecular weight
marker, Lane 2: isolate G11, Lane 3: isolate R5, lane 4: isolate Y8, lane 5: isolate
R6. The primers were designed to amplify a 0.47 kb product corresponding to an
internal fragment from within the KSα gene. Except isolate B8 and isolate G11, all
other nine isolates gave an amplicon for KS α fragment (~ 470 bps) confirming that
these isolates are indeed aromatic polyketide producers.