Plasmid DNA of Antibiotic Producing Strains of Streptomyces sannanensis Isolated from Different States in Southern India
Soil samples were collected from different states in
Southern India to isolate and characterize actinomycetes at molecular
levels through plasmid DNA and protein pattern. A total of 12 soil samples
were collected from which four strains (AP-1, KN-2, KL-3 and TN-4) were
isolated and characterized as Streptomyces sannanensis. The antimicrobial
activity of these strains was studied against gram-negative and positive-bacteria.
It was capable of producing antibiotic against gram-positive, while gram-negative
bacteria were not affected. The potential of antibiotic production of
these strains is likely to be chromosomally encoded by confirming the
detection of plasmid DNA. The strains such as KN-2 and KL-3 showed two
plasmids and the other two strains showed only one. This is a preliminary
step to correlate the chemotaxonomic relationship among the strains of
Streptomyces spp. for secondary metabolite production.
Actinomycetes population has been identified as
one of the major groups of the soil population. They have peculiar characteristics
that are transitional between bacteria and fungi and are sometimes called
as fungi-like bacteria. They are phylogenetically and chemotaxonomically
related with gram-positive bacteria with a high G+C content in their DNA
(60-70%) reported by Locci (1994). They are capable of producing spores
(Berdy, 1995), which facilitate their rapid dispersal in aquatic habitats
(Actinoplane zoospores), air/soil (Streptomycete arthrospores)
and may even ensure viability over many decades (Thermoactinomycetes).
It has been reported that they are nutritionally versatile being able
to grow both on rich substrates and on those containing a minimum or even
an apparent lack of nutrients (Wellington et al., 1992).
Actinomycetes have characteristic biological aspects
such as mycelial forms of growth that accumulates in sporulation and the
ability to produce an array of secondary metabolites many of which have
antibacterial or antifungal properties (Vasavada et al., 2006).
In fact, most antibiotics developed for human pharmaceutical use are actinomycetes
secondary metabolites, many of the being derived from Streptomyces
species (Ponmurugan et al., 2007). Complex morphological development
in the genera is phenotypically related to secondary metabolism (Ishibashi,
1992). The most promising role for secondary metabolites from actinomycetes
relies upon deference mechanisms and inhibiting other competing cells
would leave more nutrients for the survival of the secondary metabolites
producing strain. Moreover, few marine halophilic and alkaliphilic actinomycetes
have also been recently reported for their secondary metabolites production
(Kokare et al., 2004; Vasavada et al., 2006).
Many Streptomycetes carry detectable extrachromosomal
elements (plasmids) and in most cases, plasmids are present abundance
in the form of Covalently Closed Circular (CCC)-DNA, but, occasionally,
linear elements are also found (Saadoun and Blevins, 1997). The economic
importance has led to tremendous interest in the genetic aspects of antibiotic
biosynthesis by these organisms are encoded by either small or giant linear
plasmids (Stutzman-Engwall et al., 1992). So far, number of different
Streptomycetes has been investigated for plasmids and genes encode
for proteins supposedly involved in the genetic control of the production
of antibiotics (Bonjar et al., 2005). However, more detailed analyses
have shown that antibiotic biosynthetic structural genes reside mostly
on the chromosome. The findings in earlier studies indicated that the
genetic diversity in terms of plasmid DNA and protein pattern was greater
among strains of Streptomyces spp. in soil origin (Saadoun et
These recent examples from the literature highlight the
fact that despite extensive exploration of the actinomycetes for their
antimicrobial products in the past, the search for novel molecules having
unique therapeutic properties and phylogenetic relationship among strains
continues to be an active area of research (Etebarian, 2006). To keep
in mind, the present study was undertaken to isolate and characterize
the biologically diverse strains of Streptomycetes from soil samples
for the production of bioactive secondary metabolites. Studies were also
conducted to characterize the strains at molecular level in terms of extracting
MATERIALS AND METHODS
Isolation of Streptomyces spp.: Soil samples were collected
from vegetable fields at different states in Southern India such as Andhra
Pradesh (Karim Nagar), Karnataka (Mysore), Kerala (Kottayam) and Tamil
Nadu (Coimbatore) from a depth of 6-10 cm using an open-end soil borer.
A total of 12 soil samples (three samples per state/area and pooled together)
were obtained for isolation of actinomycetes by serial dilution plate
technique using casein nitrate agar (g L-1: 10 soluble starch,
0.3 casein, 2 potassium nitrate, 2 sodium chloride, 2 dipotassium hydrogen
orthophosphate, 0.05 magnesium sulphate and 0.02 calcium carbonate). These
soil samples were also subjected to analyze various parameters like pH,
total organic carbon (Walkley and Black, 1934), nitrogen (AOAC, 1990)
and available phosphorous (Jackson, 1973) and subsequently correlated
with actinomycetes distribution. Single linear regression analysis was
adopted and the data were analysed with SPSS statistical software, where
actinomycetes population density was kept as dependent variable and the
individual soil nutrient parameters were kept as independent variables.
Identification of Streptomyces spp.: There were four strains
obtained from these soils and designated as AP-1, KN-2, KL-3 and TN-4
based on the name of the state. Identification of these strains was carried
out based on morphological, physiological and biochemical tests to the
genus level following the direction mentioned in the Manual of International
cooperative project for description and deposition of Streptomyces
cultures and the method of Bergey`s manual of systemic Bacteriology (Holt,
1989). Biochemical characterization such as pigment production, starch
hydrolysis, casein hydrolysis, catalase test, oxidase test, urease test,
nitrate reduction, indole production, gelatin hydrolysis, citrate utilization
and hydrogen sulphide production were carried out to identify the name
of actinomycetes. Similarly, morphological characterization such as gram
staining, motility, nature of colony and mycelium and spore morphology
were studied. In addition, the effect of different pH and temperature
regimes on the growth of these strains was studied.
Isolation of plasmid DNA from Streptomyces spp. (Kieser, 1984):
All the four strains were grown on yeast-malt extract broth (g L-1:
3 yeast extract, 5 bacto-peptone, 3 malt extract, 10 glucose, 30 sucrose,
5 glycine and 2 mL of 2.5 M MgCl2.6H2O solution,
the latter added after autoclaving) at 28 °C under shaking at 200
rpm for 5 days. Actinomycetes-hyphae were harvested by filtration and
then washed several times in sterile distilled water. It was suspended
in 1 mL of TE buffer (40 mM Tris acetate, 2 mM EDTA, pH 7.9) and lysed
by the addition of 2 mL of freshly prepared lysis buffer (3 g SDS, 0.6
g Tris, 6.4 mL 2 N NaOH in 100 mL distilled water). It was incubated for
1 h at 55 °C and extracted with 6 mL of phenol-chloroform (1:1 V/V).
After centrifugation, the supernatant was subjected to agarose gel electrophoresis
using 0.7% agarose gels. Gels were viewed under an UV Transilluminator
(Bangalore Genei, India) and then photographed using a Gel Documentation
system (Alpha Digitoc, USA).
Screening of Streptomyces spp. for antimicrobial activity:
Each actinomycete strain was lawn cultured on casein nitrate agar
and incubated at 28 °C for 5 days. From well grown cultures, 5 mm
agar disks were prepared as described by Boyd (1995) using a sterile cork
borer and transferred to fresh lawn cultures of gram-negative organisms
such as Escherichia coli, Shigella dysentery, Pseudomonas fluorescence,
P. aeruginosa and Salmonella enteritidis and positive organisms
such as Staphylococcus aureus, Bacillus amyloliquefaciens, B. cereus,
B. megaterium and B. subtilis. After incubation at 37 °C
for 24 h, the activity was recorded by measuring the diameter of inhibition
zones for each test organism. The data obtained were subjected to analysis
of variance (ANOVA) and the significant means were segregated by Critical
Difference (CD) at 5% level of significance (Gomez and Gomez, 1984).
RESULTS AND DISCUSSION
Survey of actinomycetes diversity: In the present study, total
number of actinomycetes population present in soil samples collected from
different states in southern India indicated that it was found to be 12.5x10-3
g-1 soil dry wt. in samples collected from Andhra Pradesh followed
by Karnataka (10.9) and lesser in Tamil Nadu (6.7) regions. There was
a positive correlation between soil nutrients and population density (Table
1). The relationship was significant at 5% probability, which is coincided
with the report of Krishnakumari et al. (2006). Regression equation
was developed from the study may be useful to find out the population
density of actinomycetes of a particular locality. Actinomycetes, particularly
Streptomyces sannanensis. by virtue of their wide distribution
and antibiotic production, may participate activity in establishing the
microbiological equilibrium in soil (Moreno et al., 2003). It has
been reported that most of the isolates tend to grow in acidic soils which
is an important characteristic feature of Streptomyces spp. and
with adequate source of carbon and nitrogen present in it that enhance
the rate of multiplication (Etebarian, 2006). The survey on actinomycetes
diversity in rhizosphere soil samples collected from different districts
of Tamil Nadu was carried out by Krishnakumari et al. (2006) which
revealed the population density was found to be more in Coimbatore district
than the other districts such as Erode, Salem and Namakkal.
Characterization of actinomycetes: The results on morphological,
physiological and biochemical activity revealed all the strains of actinomycetes
belonged to Streptomyces sannanensis. They showed good sporulation
with compact, chalk-like dry colonies of different colour variations from
chalky white (AP-1) to chalky orange (KN-2). KL-3 and TN-4 strains showed
grey white and pale orange colony, respectively (Table 2).
All the strains were found to be gram-positive and showed branched mycelium
in their morphology similar to fungal characters (Holt, 1989). Aerial
mycelium was observed in KL-3 and TN-4 strains. Pigment production, hydrogen
||Population density of actinomycetes and soil nutrients
|**Significant at 1% level, *Significant at 5% level
||Characterization for identification of Streptomyces sannanensis
|++Positive reaction, -Negative reaction, +Weakly positive
||Plasmid DNA profile of Streptomyces sannanensis strains [Lane
1 - 4: Different strains of Streptomyces sannanensis (1: AP-1,
2: KN-2, 3: KL-3, 4: TN-4); M- marker DNA]
sulphide, gelatin, casein and starch hydrolysis, urease,
nitrate reduction and citrate utilization were given positive result but
catalase, oxidase, Voges-Proskaur test and indole production were negative
(Table 2). Similar results were reported recently by
several investigators (Krishnakumari et al., 2006; Vasavada
et al., 2006). The effect of pH and temperature on the growth of actinomycetes
strains were studied that revealed the optimum pH and temperature were
found to be 5-6 and 25-30 °C, respectively (Table 2).
This study may be further useful for the production antibiotics very effectively.
Molecular characterization of Streptomyces sannanensis: Lower
molecular weight CCC-DNA were detected from all the strains, but there
was no similarity between the strains in their profile (Fig.
1). The strains such as KN-2 and KL-3 exhibited two plasmids and the
other strains had only one. The molecular weight of plasmids of all the
strains was ranged between 21226 and 5148 kb. All the four strains produced
one unique band at 21226 kb (Fig. 1). Saadoun et
al. (1998) observed only CCC-DNA not linear DNA in their samples containing
the genus of Streptomyces. They were further suggesting that
||Antimicrobial activity of Streptomyces sannanensis strains
against various test organisms.
|*Average of three replicates
antibiotic production in these strains is likely to be
chromosomally encoded. Four different extraction methods of small plasmid
DNA from antibiotic-producing Streptomyces isolates and from the
positive control S. lividans, containing the pIJ702 plasmid, were
standardized. Among these, only one procedure allowed the detection of
plasmid DNA from the positive control very effectively that was the Kieser
(1984) method. This method is widely used now for the extraction of plasmid
DNA from actinomycetes (Saadoun et al., 1998).
From these results, we could discriminate all the four
strains at molecular level. However, we have to generate genetic markers
for these strains through amplification of genomic DNA using oligonucleotide
primers (RAPD analysis), as this analysis is generally applicable and
powerful for screening for bioactive principles.
Antimicrobial activity of Streptomyces sannanensis: With
the increasing use of antibiotics, the serious problem of antibiotic resistance
is gradually increasing. Therefore, intensive search for new antibiotics
is going on worldwide. Production of antibiotic as secondary metabolite
is controlled by genetic make up that imparts fullest expression and is
profoundly influenced by biotic and abiotic factors. This is substantiated
by our results presented here. All the strains of Streptomyces sannanensis
were able to produce antibiotic against gram-positive bacteria but not
against gram-negative one. Staphylococcus aureus, Bacillus amyloliguefaciens,
B. cereus, B. megaterium and B. subtilis showed positive response
while Escherichia coli, Shigella dysentery, Pseudomonas fluorescence,
P. aeruginosa and Salmonella enteritidis showed negative impact
(Table 3). Similar results were observed by Vasavada
et al. (2006) and Krishnakumari et al. (2006). The formation
of inhibition zone around the pathogenic strains is due to the production
of secondary metabolites by Streptomyces spp. Recently actinomycetes
isolated from the Sundarbans region of the Bay of Bengal, India, which
exhibited potent antimicrobial activity against gram-positive and gram-negative
bacteria, moulds, yeast and several multiple-drug resistant bacteria (Saha,
It may be concluded that out of four strains of Streptomyces
sannanensis. used, two of them such as KN-2 and KL-3 were found to
be of potential antagonists against test organisms that has the potential
to control variety of pathogenic organisms in situ.
The authors are thankful to the Director, Department
of Biotechnology, Principal and Chairman of K.S.R. Educational Institutions,
Tiruchengode, Tamil Nadu, India for providing necessary facilities and
constant encouragement to carry out this study.
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