Abstract: A Streptomyces sp. LS1-128 (closely related to S. sindenensis) isolated from the sediments of Loktak Lake, the largest freshwater lake in Eastern India, shows broad spectrum antimicrobial activity with significant inhibition zones against B. pumilus MTCC 1607 (25 mm), E. coli MTCC 739 (20 mm), B. sphaericus MTCC 2303 (19 mm), B. subtilis MTCC 121 (14 mm) and C. albicans MTCC 227 (11 mm). LS1-128 is an aerobic, gram-positive actinomycete with off-white to brown substrate mycelium with no diffusible pigment. Spores are arranged in rectiflexible chains in the aerial mycelium. It grows well in ISP2, ISP3, ISP4, ISP5, ISP6, ISP7, Streptomyces Agar and Actinomycete Isolation Agar. It can degrade starch, casein and urea and has good growth on almost all carbon sources tested including L-Arabinose, D-Fructose, D-Galactose, D-Glucose, D-Mannitol, D-Rhamnose, Salicin and D-Xylose. Based on the phenotypic and genotypic characteristics, LS1-128 was found to be most closely related to Streptomyces sindenensis (99.927% homology). The other close relatives are S. parvus, S. badius and S. globisporus). LS1-128 with promising antimicrobial activities obtained from Loktak Lake sediment has been putatively designated as Streptomyces sindenensis strain LS1-128.

INTRODUCTION

Actinomycetes especially Streptomyces sp. are rich sources of bioactive natural products with potential applications as pharmaceuticals and agrochemicals (Atta, 2009; Atta et al., 2009; Manteca et al., 2008). They are found widely in nature and in most ecological niches (Takahashi and Omura, 2003). Actinomycetes are potential sources of many bioactive compounds (Lakshmipathy and Kannabiran, 2009). They are prolific producers of secondary metabolites: antibiotics, herbicides, pesticides and anticancer agents (Atta and Ahmad, 2009; Osada, 1998; Saadoun and Gharaibeh, 2003). More than 6000 compounds are produced by Streptomyces sp. and many of them have commercial importance as anti-infectives (antibiotics, antiparasitic and antifungal agents), anticancer or immunosuppressant agents (Takahashi and Omura, 2003).

Streptomyces is the largest prokaryotic genus with 562 valid species currently (Euzeby, 2009). They are representative antibiotic producing prokaryotic group, while their morphological differentiation and genetic properties (linear chromosomes and genomic instability) have basic science interests (Locci, 1989; Chen et al., 2002).

Streptomyces sp. are widely recognized as industrially important organisms for their ability to elaborate different kinds of novel secondary metabolites (Bibb, 2005). Streptomyces sp. are prolific producers of useful bioactive compounds (Tanaka and Omura, 1990), providing 75-80% of the naturally occurring antibiotics discovered till date. They are known to elaborate a wide diversity of natural products including antibiotics, antifungal agents, plant growth factors, enzymes and enzyme inhibitors, antiparasitic, anticancer and immunomodulating agents (Berdy, 1995; Okami and Hotta, 1988; Omura, 1986; Bonjar, 2004). Streptomyces sp. or strains with novel antibiotics may still exist in nature (Okami and Hotta, 1988). They are gram positive bacteria with distinct features such as high DNA G+C content, presence of LL-Diaminopimelic acid (LL-DAP) and the absence of characteristic sugars in the cell wall (Anderson and Wellington, 2001). They also produce extensively branched substrate and aerial mycelia (Locci, 1989). They produce a wide variety of various bioactive compounds, such as antibiotics, enzymes, anticancer and agroactive compounds (Okami and Hotta, 1988; Berdy, 1995).

As exploration of the usual terrestrial sources are nearly exhausted, there is the imperative need to access various habitats such as soils, paddy fields, lake mud and water, forest and cave soils (Takahashi and Omura, 2003; Jiang and Xu, 1996; Xu et al., 1996; Kim et al., 1998).

Fig. 1:	LS1-128

Some reports say that actinomycetes of freshwater habitats can produce bioactive compounds (Okami, 1986); so more studies on aquatic actinomycetes are warranted (Rifaat, 2003). Also, there is the crying need for new antimicrobials and antifungal agents.

Actinomycetes from freshwater habitats have been relatively neglected (Goodfellow and Haynes, 1984). There is also the crying need for new antibiotics and antifungal agents.

In the ongoing search/continuing study of bioactive actinomycetes in various habitats in Manipur, part of Indo-Burma Biodiversity Hotspot (Myers et al., 2000), we had isolated a large number of actinomycetes esp. Streptomyces sp., from lake sediments of Loktak Lake, the largest freshwater lake in Eastern India (Sanasam and Ningthoujam, 2005a, b).

The aim of the present investigation was to screen a subset of our actinomycete collection for antagonistic activities against bacteria and fungi of medical and agricultural importance and characterize the bioactive strains. During this study, we found a strain, LS1-128 (Fig. 1), with interesting broad spectrum activities against selected bacterial and fungal pathogens. This study deals with bioactivity of the strain and polyphasic characterization of the antagonistic strain to establish its taxonomic position.

MATERIALS AND METHODS

Media and reagents: Microbial growth media were obtained from Himedia (Mumbai, India) or prepared in the Lab according to standard protocols. All other reagents and chemicals were of the highest grade available.

Loktak Lake: Loktak Lake is one of the largest freshwater lakes in Eastern India. It has been covered under Ramsar convention in 1990 (Ningombam and Bordoloi, 2007).

The lake’s ecology is in precarious state due to shallowing by siltation and pollution by incoming streams and rivers and from human activites and agricultural runoff. Its condition worsened after launching of a hydroelectric project in the lake about 30 years ago.

Sampling: Sediment samples were collected from the bottom of Loktak Lake at various depths and stored in refrigerator till processing. They were processed soon after collection.

Isolation of lake actinomycetes: The strain was isolated from sediments of Loktak Lake, the largest freshwater lake in Eastern India using Starch Casein Nitrate Agar (Kuster and Williams, 1964). It is maintained on Bennett’s agar slants (Jones, 1949) at 28 and 4°C.

Indicator organisms: The test organisms Bacillus pumilus, B. sphaericus, B. subtilis, E. coli and Candida albicans were procured from Microbial Type Culture Collection (MTCC), Institute of Microbial Technology (IMTECH), Chandigarh, India. Pseudomonas aeruginosa DN1 is a lab isolate and part of the collection of Microbial Biotechnology Research Lab (MBRL), Manipur University (MU), Canchipur, India.

Antimicrobial screening: The antimicrobial assay was done by Bauer et al. (1966) method against S. aureus MTCC 96, B. subtilis MTCC 121, B. pumilus MTCC 1607, B. sphaericus MTCC 2303, E. coli MTCC 739, P. aeruginosa DN1, C. albicans MTCC 227 and A. niger MTCC 1344 obtained from Microbial Type Culture Collection (MTCC), IMTECH, Chandigarh, India, except for DN1 which is a lab collection.

Phenotypic characterization: The morphology of the spore chain and the spore surface were examined by light microscopy of 14 days old culture on inorganic salts-starch agar. The cultural properties of the strain were evaluated according to the guidelines of the International Streptomyces Project (ISP) as described by Shirling and Gottlieb (1966).

Cell wall chemotype analysis: The isomeric form of Diaminopimelic acid (DAP) in the whole cell hydrolysates were analysed by TLC (Staneck and Roberts, 1974).

Genotypic characterization: 16S rDNA amplification and sequencing were carried out as described by Kim et al. (1998). The almost complete 16S rDNA sequence was submitted to EzTaxon server (Chun et al., 2007), which contains manually curated databases of type strains of prokaryotes for sequence analysis. Related strains were selected for alignment by CLUSTAL W program and were done phylogenetic analysis according to the neighbor-joining method (Saitou and Nei, 1987) using the MEGA version 4.1 (Tamura et al., 2007). To determine the support of each clade, bootstrap analysis was performed with 1000 replications (Felsenstein, 1985).

RESULTS AND DISCUSSION

Strain LS1-128 Fig. 1 shows significant activity against 5 of the 8 test organisms tested (Table 1).

Physiological and biochemical characteristics: The phenotypic characteristics of the strain LS1-128 is shown in Table 2.

Cell-wall chemotype analysis: Whole-cell hydrolysates contained LL-DAP as diagnostic acid of the cell-wall peptidoglycan, establishing the strain to be belonging to Streptomyces.

Phylogenetic analysis: The almost-complete 16S rRNA gene sequence (1363 nt) of strain LS1-128 shows that the organism belongs to the genus Streptomyces. A phylogenetic tree was constructed for LS1-128 based on the 16S rDNA sequences of the strain and other related Streptomyces sp. The strain shows closest affinity with Streptomyces sindenensis (Fig. 2) with sequence homology of 99.927%.

Description of Streptomyces sp. strain LS1-128: Strain LS1-128 produced rectiflexibilis spore chains in the aerial mycelium. The colour of the substrate mycelia was off-white to brown. No diffusible pigments were produced. Strain LS1-128 grew well on yeast extract malt extract agar (ISP2-HiMedia), oatmeal agar (ISP3-HiMedia), inorganic salts/starch agar (ISP4-HiMedia), glycerol asparagine agar (ISP5-HiMedia), peptone/yeast extract iron agar (ISP6-HiMedia), tyrosine agar (ISP7-HiMedia), Streptomyces Agar (HiMedia) and Actinomycete Isolation Agar (HiMedia). Starch, Casein and urea are degraded. Good growth are found on almost all carbon sources tested including L-Arabinose, D-Fructose, D-Galactose, D-Glucose, D-Mannitol, D-Rhamnose, Salicin and D-Xylose. Whole cell hydrolysates contained LL-DAP as diagnostic acid of the cell-wall peptidoglycan. Cell wall fatty acid analysis shows that it contains glucose, galactose and ribose.

Table 1:	Antimicrobial activity profile of LS1-128

Table 2:	Characteristics of LS1-128
+: Positive; -: Negative; w: Weakly positive

Fig. 2:	Phylogenetic analysis of LS1-128 with 10 nearest homologs

However, whether LS1-128 is a novel Streptomyces sindenensis strain or a novel species could only be ascertained by DNA-DNA hybridization and other molecular studies with related strains from the sub-clade. There are limited reports on bioactivity of Streptomyces sindenensis. For example, Praveen et al. (2008) reported actinomycin-D production by a new isolate of Streptomyces sindenensis but their strain was obtained from soil sample collected from steel plant effluents in Uttar Pradesh, India.

Zarandi et al. (2009) obtained a strain of S. sindenensis from soils in Karman Province, Iran with biocontrol activity against Magnaporthe oryzae (causal agent for blast disease in rice). To the knowledge, this is the first report of a bioactive strain of Streptomyces sindenensis recovered from lake sediments.

Actinomycetes play major roles in C cycle in aquatic habitats and wetlands, due to their ability to grow at the low C concentrations and degrade recalcitrant organic matter (Kuznetsov, 1970). Therefore, they form promising sources for the survey of actinomycetes as they are well known as excellent decomposers.

However, exploration of actinomycete diversity in lakes, rivers, ponds, wetlands and other aquatic ecosystems are relatively still neglected. There are as yet scant reports on lake actinomycetes especially those from freshwater lakes. However, some groups have reported the predominance of Streptomyces in freshwater lake sediments and of Micromonospora in lake water (Terkina et al., 2002, 2006). Similar findings have also been reported for marine sediments (Jensen et al., 1991, 2007). Of late, novel drugs such as salinosporamide have been discovered from marine actinomycetes such as Salinispora (related to Micromonospora) (Jensen et al., 2007).

Terkina et al. (2002) found that Streptomyces and Micromonospora were the predominant genera in Lake Baikal using starch-containing agar media who observed that lake water was dominated by Streptomyces (66% of all water isolates) and sediments by Micromonospora (59% of all sediment isolates).

Rifaat (2003) also found that, of 114 strains from River Nile, Streptomyces were prevalent in water and Micromonospora in sediments and several Streptomyces strains had antimycotic activity.

Freshwater habitats have been recently stressed as promising sources of bioactive metabolites (Cross, 1981). Terkina et al. (2002, 2006) reported that several lake Baikal actinomycetes inhibited the growth of several pathogens including antibiotic-resistant microbes. Elliah et al. (2002) observed that Streptomyces strains from Krishna river sediments in India had antibacterial and antifungal activities. Rifaat (2003) in his studies of actinomycetes from River Nile in Egypt, reported that several Streptomyces strains had significant antimycotic activity. New actinomycete strains or species have also been recovered from lakes and other aquatic environments. For example, Lango et al. (1999) identified Streptomyces galbus from sediment of La Caldera in Spain.

LS1-128 was isolated from sediments of Loktak Lake in Manipur, India. We had obtained 172 lake sediment actinomycetes using SCNA medium. A majority of them were found to be Streptomyces and nocardioform actinomycetes. This differs from the findings of Terkina et al. (2002, 2006) who observed predominance of Micromonospora in lake Baikal sediments and Streptomyces in lake Baikal water. Similar findings were also found by Rifaat (2003). But this may be a reflection of the trophic status of the contrasting lakes; whereas Lake Baikal is an oligotrophic lake, Loktak Lake is highly eutrophic, polluted by inlet streams and rivers loaded with wastes and agricultural runoff.

Nocardioform actinomycetes have earlier been reported from aquatic habitats, with their presence as possibly linked with anthropogenic activities as suggested by some researchers (Rowbotham and Cross, 1977; Yamamura et al., 2003).

It is not surprising that we got a large number of actinomycete isolates from Loktak lake sediments as actinomycetes comprise up to 30% of the microbial population in lakes, the largest fraction that are culturable being Micromonospora and streptomycetes (Terkina et al., 2002; Sponga et al., 1999; Takizawa et al., 1993; Jiang and Xu, 1996; Jensen et al., 1991).

The present study indicates that Loktak Lake can be a promising source of bioactive metabolites and of novel strains or species of actinomycetes. Detailed characterization of LS1-128 and its bioactive metabolite (s) and screening of our remaining lake actinomycete isolates will now be the target of the further studies.

ACKNOWLEDGEMENTS

This study was supported by a grant from DST&IT, Govt. of Manipur. We gratefully acknowledge the generous help of Prof. Rup Lal, Head, Department of Zoology, Delhi University for extending the services of the DNA sequencing facility.