Characterization of Streptomyces strain SLO-105 isolated from Lake Oubeira sediments in North-East of Algeria

Abstract

A microbial strain, SLO-105, isolated from Lake Oubeira sediment was screened for its antimicrobial activity against pathogenic bacteria and fungi. The strain showed broad-spectrum antibacterial activity against Gram-positive bacteria Staphylococcus aureus MRSA, Bacillus subtilus, Micrococcus leutus, Streptococcus fecalis and fungi Aspergillus niger and Rodotorulla mucilaginosa. However, no activity of the strain was observed against Gram negative bacteria Escherichia coli and Pseudomonas aeruginosa as well as on fungi Candida albicans. Analysis of 16S rDNA sequence and the morphological and physiological characteristics of the strain suggested that the isolate belonged to Streptomyces genus.

Full text

Page 1

African Journal of Biotechnology Vol. 8 (22), pp. 6332-6336, 16 November, 2009
Available online at http://www.academicjournals.org/AJB
ISSN 1684–5315 © 2009 Academic Journals





Full Length Research Paper

Characterization of Streptomyces strain SLO-105
isolated from Lake Oubeira sediments in North-East of
Algeria

Houda Morakchi1,2*, Adel Ayari1, Mira Taok2, Djamila Kirane1 and Nelly Cochet2

1Applied Microbial Laboratory, Department of Microbiology and biochemistry, Badji Mokhtar University, P. O. Box 12.
23000 Annaba, Algeria.
2University of Technology of Compiegne. BP 20 529. 60 205. Compiegne, France.

Accepted 29 September, 2009

A microbial strain, SLO-105, isolated from Lake Oubeira sediment was screened for its antimicrobial
activity against pathogenic bacteria and fungi. The strain showed broad-spectrum antibacterial activity
against Gram-positive bacteria Staphylococcus aureus MRSA, Bacillus subtilus, Micrococcus leutus,
Streptococcus fecalis and fungi Aspergillus niger and Rodotorulla mucilaginosa. However, no activity
of the strain was observed against Gram negative bacteria Escherichia coli and Pseudomonas
aeruginosa as well as on fungi Candida albicans. Analysis of 16S rDNA sequence and the
morphological and physiological characteristics of the strain suggested that the isolate belonged to
Streptomyces genus.

Key words: Streptomyces, rDNA analysis, micro-morphology, physiology, antimicrobial.


INTRODUCTION

New microbial metabolites, particularly antibiotics, are
permanently needed due to the increase of resistant
pathogens, evolution of novel diseases and toxicity of
currently used compounds (Hakvag et al., 2008; Jensen
et al., 2005). Approaches to the discovery of new anti-
biotics are generally based on screening of naturally
occurring microorganisms like Actinomycetes, (Donadio
et al., 2002) in different environmental samples.
Actinomycetes represent a large group of Gram-
positive filamentous bacteria that currently comprises
over forty genera and hundreds of species among which
is the important Streptomyces genus (Watve et al., 2001).
Members of this group have been isolated from different
soils, plant materials, waters and marine sediments
(zaitlin et al., 2003; Jensen et al., 1991). They are able to
produce a wide range of molecules with broad spectrum
of activities, that is, antibacterial, antifungal, antitumural,
antiparasitic and antiviral (Atta and Ahmad, 2009;
Naeimpoor and Mavituna, 2000).



*Corresponding author. E-mail: hmorakchi@yahoo.fr.
The present work described the cultural and physiological
characteristics as well as the antibacterial activity of an
Actinomycete strain, SLO-105, isolated from Lake
Oubeira sediment.


MATERIALS AND METHODS

Strain isolation and storage

The strain SLO-105 was isolated from sediment sample collected
from the Oubeira Lake. One gram of sample was diluted (10-1 to 10-
6) with sterile water and spread on glycerol-asparagines agar (L-
asparagine: 0.1%, glycerol: 1%, K2HPO4: 0.1%, 1 ml of trace salts
solution: FeSO4.7H2O: 0.1%, MnCl2.4H2O: 0.1%, ZnSO4.7H2O:
0.1%, pH 7.4) containing 50 µg/ml of cycloheximide in order to mini-
mize fungal contamination. Plates were incubated for 2 weeks at
30° C (Matsukawa et al., 2007). The strain SLO-105 was confirmed
by colony morphology as actinomycetes and then purified by streak
plate technique (Thakur et al., 2007). For long storage for further
use, spores stocks of the isolate were prepared as described by
Hopwood et al. (1985). The spore suspension was analyzed by a
spectrophotometer at 450 nm UVI light XS SECOMAM (Nova
Analytics company) and the absorbance was adjusted to 0.05
(about 107 CFU/ml) by adding distilled water. The same volume of
glycerol was added to the spore suspension and aliquots of 1 ml

Page 2

were stored in cryotubes at -20°C and – 80°C (Elmahdi et al., 2003;
Hopwood et al., 1985).


Morphological characteristics

The micro morphology of the strain was carried out using a scan-
ning electron microscope Philips XL 30 ESEM-FEG (Philips Optique
Electronique, Limeil Brévannes, FRANCE). The sample used for
observation has been cultured for 14 days on Yeast malt glucose
agar (Bacto-yeast extract: 4.0 g/l, Bacto-malt extract: 10 g/l, glu-
cose: 4.0 g/l, distilled water: 1000 ml, agar: 20 g/l. pH 7.3)
(Boudjellel et al., 2006).


Cultural, physiological and biochemical characteristics

The morphological and cultural characteristics of strains SLO-105
were made according to the guidelines of the International Strep-
tomyces Project (Shirling and Gottlieb, 1966). The cultural aspect of
the pure strain was studied on different ISP medium after 14 days
incubation at 28° C. Colors were determined according to ISCC-
NBS color charts (Kenneth, 1958).
Carbohydrates utilization was determined by growth on carbon
utilization medium (ISP9) supplemented with 1% carbon sources
(Pridham and Gottlieb, 1948) and incubate at 30° C. Melanin pro-
duction was observed on peptone yeast iron agar and tyrosine agar
(Pridham et al., 1957).
Hydrolysis of gelatin was evaluated according to Waksman
(1961) method. Degradation of adenine, guanine and L-Tyrosine
were determinate using Goodfellow (1971) medium. Hydrolysis of
casein and starch were evaluated by using the media of Gordon et
al. (1974). Tween 80 was observed on Sierra and Greuell medium
(1957), lecithinase and chitinase activity were evaluated respec-
tively on egg-yolk medium (Nitsch and Kutzner, 1969) and
Powdered Chitin Agar (Hsu and Lockwood, 1975).


Antibiotic bioassay

The antimicrobial activity of strain SLO-105 was evaluated on solid
media by double layers method (Mellouli et al., 2003; Hu et al.,
2000). After incubation of the strain for 8 days at 30°C, plates were
covered by 4 ml of top agar previously seeded with 50 µl of fresh
suspension of the test organisms. Plates were incubated 24 h at
37° C for bacteria and 3 - 5 at 30° C for fungi.
The test organisms were Escherichia coli ATCC 25922, Pseudo-
monas aeruginosa ATCC 9027, Bacillus subtilus ATCC 6633,
Micrococus luteus ATCC 4698, Streptococcus fecalis ATTC 49332,
the pathogenic methicillin resistant Staphylococcus aureus MRSA
ATCC 43300, Aspergillus niger ATCC 16404, Candida albicans
ATCC 10231 and Rodotorulla mucilaginosa ATCC 9449.


DNA isolation

A loop of culture of strain cultured on solid medium (ISP2) was
suspended in 0.5 ml of Tris-EDTA buffer (10 mM Tris, 1 mm EDTA,
pH 8). 50 ?l of lysozyme solution (100mg/ml) was added to the
buffer. After overnight incubation at 37° C, 70 ?l of a solution in 10%
SDS and 6 ?l of proteinase K (10 mg/mL) were added to the buffer
and the solution was incubated at 65° C for 10 min. 500 ?l of
chloroform-isoamyl alcohol (24:1) were added to the solution and
vortexed for 10 s, then centrifuge at 3000 g for 10 min. DNA was
precipitated with 600 ?l of isopropanol at -20° C for 30 min and then
centrifuged at 15,000 rpm for 10 min. The pellet was washed 2
times with ethanol, dried at room temperature and then resus-
pended in 10 ?l of 1X TE buffer and stored at -20° C (Valenzuela-
Morakchi et al. 6333



Tovar et al., 2005)


Amplification and sequencing of the 16S rRNA gene

The amplification of the 16S gene was conducted using the kit
Accuprime Taq and two primers 63F (CAGGCCTAACACATG
CAACTC) and 1387r (GGGCGGGTGTACAAGGC). The PCR mix-
ture consisted of 5 ?l of 10X PCR buffer (final concentration: 1X)
0.6 ?l primer 16S-63F, 0.74 ?l primer 16S-1387r, 5 units / ?l of Taq
Polymerase and 1 ?L genomic DNA to a final volume of 50 ?l.
PCR was carried out in a thermocycler (Eppendorf mastercycler
gradient) using a program that consisted of an initial denaturation at
94° C for 2 min, then 30 cycles of elongation (92° C for 30 s, 50° C for
30 s and 72° C for 1 min). The last step was performed at 72° C for 5
min. Two blanks containing all components of the reaction mixture
except the DNA sample are routinely used as controls (Hu et al.,
2002). The PCR products were analyzed by agarose gel
electrophoresis.
The sequencing was performed (Genoscreen, France) using the
BigDye Terminator v3.1 Cycle Sequencing Kit and the 3730xl
sequencer (Applied Biosytems, Fermont, Calif.). The 16S DNA
sequences obtained was compared with sequences deposited in
GenBank genomic bank using the BLAST program (Version 2.0)
available on the site http://www.ncbi.nlm.nih.gov/Blast. Sequence
data for related species were retrieved and aligned using the
Clustal X program. The phylogenetic tree was constructed via the
neighbor-joining algorithm (Saitou and Nei, 1987) using the soft-
ware MEGA 4.0.2 (Tamura et al., 2007). The sequence data was
deposited in the Genbank Database, under the accession number
GQ371155.


RESULTS AND DISCUSSION

The strain SLO-105 was isolated from lake Oubeira
sediment situated in The National Park of El Kala (North-
East of Algeria) which covers an area of 78 400 ha and
contains a set of wetlands of high ecological value (Van
Dijk and Ledant, 1983). The Lake Oubeira is a fully
protected area and include a large wetland territory
(76,438 ha). It has been stated of International Impor-
tance under the Ramsar Convention.
The scanning electron micrograph of the strain SLO-
105 (Figure 1) revealed that the aerial mycelia were
monopodially branched with compact sporophore. The
spores had a smooth surface, no fragmentation of
substrate mycelia was observed and no sclerotia, spo-
rangia, or flagellated spores were formed.
The cultural characteristics of the strain listed in Table
1 showed moderate to good growth on most media. Typi-
cally, the colony was elevated, spreading and covered
with vivid orange yellow aerial mycelium. The reverse
side showed a deep orange yellow color and the strain
produce a vivid yellow pigment on most media except on
the ISP5.
The morphological and cultural characteristics of the
isolate were compared with known Actinomycetes
species described in Bergey’s manual of systematic
bacteriology and they suggested that SLO-105 strain
belong to Streptomyces genus. This was confirmed by
16S rDNA gene sequence analyzed. The alignment of
this sequence with the 16S rDNA retrieved from the

Page 3

6334 Afr. J. Biotechnol.





Figure 1. Scanning electron micrographs of the spore chains of strain SLO-105 grown on Yeast malt glucose
agar at 30°C for 14 days.



Table 1. Cultural characteristics of strain SLO-105.

Medium
Tryptone yeast extract (ISP1)
Yeast-malt-glucose (ISP2)
Oatmeal (ISP3)
Inorganic salts starch (ISP4)
Glycerol asparagines (ISP5)
Growth
Poor
Good
Moderate
Moderate
Moderate
Aerial mycelium
Vivid Orange Yellow
Vivid Orange Yellow
Light Gray
Vivid Orange Yellow
Light Orange Yellow
Substrate mycelium
deep orange yellow
deep orange yellow
deep orange yellow
deep orange yellow
light orange yellow
Soluble pigment
Vivid Yellow
Vivid Yellow
Vivid Yellow
Vivid Yellow
None



genbank database revealed a high degree of similarity
(98-99%) with Streptomyces 16S rDNA gene. Based on
phylogenetic analysis (Figure 2) the strain was highly
related to Streptomyces galbus strain DSM 40089 with
99% of similitude.
The physiological and biochemical characteristics of
the isolate shown in Table 2 indicated that SLO-105
strain have the ability to utilize all tested carbon sources.
The isolate produce melanin pigment when cultivated on
either ISP6 or ISP7 and produced hydrogen sulfide. It was
also able to degrade adenine, tween 80 and liquefy
gelatin but not able to degrade casein, starch, guanine
and L-tyrosine. The strain shown a lecithinase activity on
egg-yolk agar but no chitinase activity was observed.
The active metabolites produced by SLO-105 strain
exhibited various degrees of activities against tested
organisms (Table 3). Antimicrobial activities expressed as
the inhibition zone diameter showed that highest activities
were observed on Gram positives bacteria S. fecalis (76
mm), M. luteus (72 mm), S. aureus MRSA (60 mm) and
was also active against A. niger (45 mm) and R. mucila-
ginosa (55 mm). No activity was observed on fungi

Page 4

Morakchi et al. 6335










Figure 2. The phylogenetic position of SLO-105 strain among neighboring species.



Table 2. Physiological and biochemical characteristics of
strain SLO-105.

Characteristics Strain SLO-105
??
Carbon utilization
Glucose
Arabinose
Fructose
Sucrose
Xylose
Lactose
Mannitol
Production of melanin pigment
+
+
+
+
+
+
+
??
peptone-yeast extract-iron agar ( ISP6)
Tyrosin agar (ISP7)
Degradation activity
+
+
??
Adenine
Guanine
L-Tyrosine
Tween 80
Starch
???????
+
-
-
+
-
??
Hydrogen sulfide production
Hydrolysis of casein
Liquefaction of gelatin
Lecithinase production
Chitinase production



Candida albicans and Gram negative tested bacteria.
In this work, we have shown the ability of the strain
SLO-105 to produce antimicrobial compounds against
+
-
+
+
-
Table 3. Antibacterial activity of SLO-105 strain on solid
media.

Organism test Inhibition zone diameter (mm)
Bacteria
B. subtilis
E. coli
P. aeruginosa
S. aureus MRSA
M. luteus
S. fecalis
Fungi
C. albicans
A. niger
R. mucilaginosa
68
-
-
60
72
76

-
45
55



microorganisms, especially the multiple antibiotic resis-
tant Gram positive bacteria MRSA ATCC 43300 and the
fungi A. niger and R. mucilaginosa. Further investi-
gations on the extraction, purification and characteriza-
tion of the active compound are currently in progress.


REFERENCES

Atta HM, Ahmad MS (2009). Antimycin-A Antibiotic Biosynthesis
Produced by Streptomyces
Fermentation, Purification and Biological Activities. Aust. J. Basic
Appl. Sci. 3(1): 126-135.
Boudjelle H, Bouti K, Zitouni A, Mathieu F, Lebrihi A, Sabaou N (2006).
Taxonomy and chemical characterization
Streptosporangium Sg 10 isolated from a Saharan soil. Microbiol.
Res. 161: 288-298.
Donadio S, Monciardini P, Alduina R, Mazza P, Chiocchini C,
Cavaletti L, Sosio M, Puglia AM (2002). Microbial technologies for the
discovery of novel bioactive metabolites. J. Biotechnol. 99: 187-198.
Elmahdi I, Baganz F, Dixon K, Harrop T, Sugden D, Lye GJ (2003). pH
control in microwell fermentations of S. erythraea CA340: influence
on biomass growth kinetics and erythromycin biosynthesis. Biochem.
Eng. J. 16: 299-310.
Goodfellow M (1971). Numerical taxonomy of some nocardioform
Sp. AZ-AR-262: Taxonomy,
of antibiotics of

Page 5

6336 Afr. J. Biotechnol.



bacteria. J. Gen. Microbiol. 93: 33-80.
Gordon RE, Barnett DA, Handehan JE, Pang CH (1974). Nocardia
coeliaca, Nocardia autotrophica and Nocardia Strain. Int. J. Syst.
Bacteriol. 24: 54-63.
Hakvag S, Fjærvik E, Josefsen KD, Ian E, Ellingsen TE, Zotchev SB
(2008). Characterization of Streptomyces spp. Isolated from the Sea
Surface Microlayer in the Trondheim Fjord, Norway. Mar. Drugs, 6:
620-635.
Hopwood DA, Bibb MJ, Chater KF, Kieser T, Bruton CJ, Kieser HM,
Lydiate DJ, Smith CP, Ward JM, Schrempf H (1985). Genetic
Manipulation of Streptomyces: A Laboratory Manual. John Innes
Foundation, Norwich, United Kingdom.
Hsu SC, Lockwood JL (1975). Powdered Chitin Agar as a Selective
Medium for Enumeration of Actinomycetes in Water and Soil. Appl.
Microbiol. 29: 422-426.
Hu HB, Lackovich JK, Lee JE (2002). High specificity and robustness of
AccuPrime™ Taq provides the ideal tool for demanding miniaturized,
multiplex, and high-throughput PCR. Focus, 24(2): 11-13.
Hu ZY, Liou SZ, Huang YJ, Su WJ (2000). A rapid screening assay for
insecticidal antibiotic produced by marine actinomycetes. Mar. Sci.
Bull. 19: 36-41.
Jensen PR, Mincer TJ, Williams PG, Fenical W (2005). Marine
actinomycete diversity and natural product discovery. Antonie van
Leeuwenhoek 87: 43-48.
Jensen PR, Dwight R, Fenical W (1991). Distribution of actinomycetes
in near-shore tropical marine sediments. Appl. Environ. Microbiol. 57:
1102-1108.
Kenneth LK (1958). Prepared research paper RP 2911, Central
Notations for the Revised ISCC-NBS color name blocks. J. Res.
NBS, 16: p. 427.
Matsukawa M, Nakagawa Y, Limura Y, Hayakawa M (2007). A New
Enrichment Method for the Selective Isolation of Streptomycetes from
the Root Surfaces of Herbaceous Plants. Actinomycetologica 21: 66-
69
Mellouli L, Ben Ameur-Mehdi R, Sioud S, Salem M, Bejar S (2003).
Isolation, purification and partial characterization of antibacterial
activities produced by a newly isolated Streptomyces sp. US24 strain.
Res. Microbiol. 154: 345-352.
Naeimpoor F, Mavituna F (2000). Metabolic Flux Analysis in
Streptomyces coelicolor under Various Nutrient Limitations. Metab.
Eng. 2: 140-148.
Nitsch B, Kutzner HJ (1969). Egg-Yolk agar as diagnostic medium for
Streptomycetes. Experientia, 25: 113-116.

































Pridham TG, Gottlieb D (1948). The utilization of carbon compounds by
some actinomycetes as an aid for species determination. J. Bacteriol.
56(1): 107-114.
Pridham TG, Anderson P, Foley C, Lindenfelser LA, Hesselting CW,
Benedict RG (1957). A section of media for maintenance and
taxonomic study of Streptomycetes. Antibiotics Ann. pp. 947-953.
Saitou N, Nei M (1987). The neighbor-joining method for reconstructing
phylogenetic trees. Mol. Biol. Evol. 4: 404-425.
Sierra G, Greuell EHM (1957). Studies on bacterial esterases: (i)
differentiation of a lipase and two di-esterases during the growth of
Pseudomonas aeruginosa and some observations on growth and
esterase inhibition; (ii) paper electroporosis of the esterases of
Pseudomonas aeruginosa; (iii) influence of the composition of the
culture media on the production of di-esterase and lipase of
Pseudomonas aeruginosa. Antonie van, Leewenhoek, 23: 241-278.
Shirling EB, Gottlieb D (1966). Methods for characterization of
Streptomyces species. Int. J. Syst. Bacteriol. 16: 313-340.
Tamura K, Dudley J, Nei M, Kumar S (2007). MEGA 4: Molecular
Evolutionary Genetics Analysis (MEGA) software version 4.0. Mol.
Biol. Evol. 24: 1596-1599.
Thakur D, Yadav A, Gogoi BK, Bora TC (2007). Isolation and screening
of Streptomyces in soil of protected forest areas from the states of
Assam and Tripura, India, for antimicrobial metabolites. J. med.
Mycol. 17: 242-249
Van Dijk G, Ledant JP (1983). La valeur ornithologique des zones
humides de I'Est algérien Biol. Cons. pp. 215-226.
Valenzuela-Tovar JF, Contreras-Perez C, Shibayama-Hernandez H,
Chavez-Gonzalez L, Vazquez-Chacon CA, Olivera-Diaz H (2005).
Biochemical identification and molecular characterization (PCR-
RFLP) of Nocardia isolates from sputum. Arch. Med. Res. 36: 356-
361.
Waksman SA (1961). The actinomycetes. Classification, Identification
and Description of Genera and Species, vol. 2. Williams and Wilkins
Company, Baltimore, USA, p. 327.
Watve MG, Tichoo R, Jog MM, Bhole BD (2001). How many antibiotics
are produced by the genus Streptomyces. Arch. Microbiol. 176: 386-
390.
Zaitlin B, Watson SB, Ridal J, Satchwill T, Parkinson D (2003).
Actinomycetes in Lake Ontario: Habitants and Geosmin and MIB
production. Am. Water Works Assoc. J. 95(2): 113-118.