Characterization of Indigenous Copper-Resistant Streptomycetes from Chickpea (Cicer Arietinum L.) Fields

Ann Microbiol (2010) 60:605–614 DOI 10.1007/s13213-010-0095-3

ORIGINAL PAPER

Characterization of indigenous copper-resistant Streptomycetes from chickpea (Cicer arietinum L.) fields

Arvind K. Yadav & Alok K. Srivastava & Mahesh S. Yandigeri & Sudhanshu K. Kashyap & Dinesh R. Modi & Dilip K. Arora

Received: 25 February 2010 /Accepted: 25 June 2010 /Published online: 23 July 2010 # Springer-Verlag and the University of Milan 2010

Abstract A total of 432 morphotypes of Streptomycetes Streptomyces sp. (S160 and S161) and Streptomyces were isolated from rhizospheric and non-rhizospheric soils hygroscopicus (S164), respectively. of chickpea (Cicer arietinum L.) which showed significant variation in population count. The generation time at Keywords Actinomycetes . Copper resistance . Selfish different Cu2+ concentrations (0.5, 0.75 and 1.0 mM), were genes . Streptomyces recorded for S160 as (1.2, 6.0 and 14.4 h), S161 (7.2, 12.0 and 24.0 h), and S164 (4.8, 7.2 and 16.8 h), respectively. Qualitative results showed 100, 62 and 8% of non- Introduction rhizospheric isolates tolerated 0.075, 0.264 and 0.628 mM of Cu2+, respectively, while rhizospheric isolates showed Actinomycetes, the Gram-positive filamentous sporulating tolerance of 0.264 mM CuSO4. Semi-quantitative assay bacteria with high G+C content (60–70 mol %), are important showed that the rhizospheric strain S160 has the ability to sources of antibiotics (>50%) and enzymes (Edwards 1993; withstand 3.14 mM, while non-rhizospheric isolates S161 Gharaibeh et al. 2003; Weber et al. 2003). On the basis of and S164 were able to tolerate up to 3.14 and 1.88 mM morphological and chemotaxonomical criteria, they have

CuSO4, respectively. Acid-digested pellet of S160 strain been grouped into different genera (Wang et al. 1999). indicated 68% reduction in copper residual concentration, Actinomycetes with type I cell wall, complex life cycle, confirming copper accumulation capacity in Streptomyces. having three stages of differentiation are classified as The ability of growth of these strains in the presence of Streptomycetes within the family Streptomycetaceae Cu2+ was co-induced by the presence of 3.1, 2.8 and 2.1 kb (Stackebrandt et al. 1997). Screening and isolation of plasmid, respectively. Plasmids transformation into wild promising actinomycetes with potential antimicrobial strain resulted in changes in metal resistance ability along compounds is still a thrust area of research and it is with appreciable changes in resistance to antibiotics. suggested that the exploration of new areas and habitats Cultures were identified by 16S rDNA sequencing as played a vital role in the search for new microbes and novel metabolites (Horinouchi 2002;Ohnishietal.2005). Streptomycetes are the most abundant group of bacteria in soils (70%) and show primary biodegradative activity, secreting a range of extracellular enzymes that * : : : A. K. Yadav ( : ) A. K. Srivastava M. S. Yandigeri allow them to metabolize recalcitrant molecules (Kieser S. K. Kashyap D. K. Arora et al. 2000). This morphological and metabolic versatility National Bureau of Agriculturally Important Microorganisms (ICAR), Kusmaur, P.B. No.6, Mau Nath Bhanjan 275101, gives them a great potential to perform in bioremediation Uttar Pradesh, India processes, including metal recovery (Ravel et al. 1998). e-mail: [email protected] They have been reported for their role in soil ecology by decomposing organic matter and thus contributing to soil D. R. Modi Department of Biotechnology, B. B. R. Ambedkar University, fertility (Molla et al. 1984; Pasti et al. 1990;Vargasetal. Lucknow 226025, India 2009). Copper is an essential element required by all 606 Ann Microbiol (2010) 60:605–614 organisms as it is an important co-factor for many methods (Rowell 1994; American Public Health Associa- enzymes involved in vital cellular processes (Albarracin tion 1980) (Table 1). et al. 2005; Tsivkovskii et al. 2003). It cannot be destroyed and tends to accumulate in the organisms at higher tropic Isolation and strain characterization levels and directs toxicity to vertebrates within a range of 100–1,000mgL−1 (Georgopoulus et al. 2002), and it has Streptomycetes were isolated in minimal agar (MA) −1 been used in copper-based fungicides on agricultural crops medium containing (g L ): L-asparagine, 0.5; K2HPO4, (Fairbrother et al. 1999). Preliminary information is 0.5; MgSO4.7H2O, 0.2; FeSO4.7H2O, 0.01; glucose, 10; available on copper resistance mechanisms in Gram- agar, 15; supplemented with 0.1 mM CuSO4 (pH 7). The negative bacteria such as Escherichia coli and Pseudomonas medium was supplemented with nalidixic acid and sp. (Munson et al. 2000). Nevertheless, specific information cycloheximide (10 mg mL−1 each) to inhibit Gram- pertaining to the mechanisms involved in the resistance to negative bacteria, some Gram-positive bacteria, and fungi, copper by actinomycetes is not yet known (Erardi et al. respectively (Ravel et al. 1998). Plates were incubated at 1987). The antagonistic impacts of actinomycete species on 30±2°C for 7 days and colonies were purified by streaking pathogenic fungi are well known and a few species have on agar medium without antibiotics. been used as biological control agents (El-Shanshoury et al. Chemotaxonomic characterization, cell wall preparations, 1966; El-Tarabily et al. 1997; Jones and Samac 1996; Yuan and whole cell analysis were carried out by inoculating the and Crawford 1995), but a study pertaining to copper screened isolates in ISP-2 broth and harvested by centrifuga- tolerance along with biological control ability has not been tion and finally washing in sterile distilled water. Hydrolysate attempted. The objective of the present study was to for di-amino pimelic acid (DAP) analysis was prepared using decipher the distribution pattern and characterize copper- standard methods (Becker et al. 1965), followed by ascending resistant Streptomycetes from rhizospheric and non- rapid thin-layer chromatography (cellulose and silica coated rhizospheric soils of chickpea (Cicer arietinum L.) fields TLC plates 5 × 20 cm; Merck, India), carried out by spotting of different soil types, taxonomically characterize promising with 5 μL of the hydrolysate and developed for 2–2.5 h in n- copper-tolerant isolates, on the basis of morphological and butanol: acetic acid: water (4:1:1) for DAP and chloroform: biochemical parameters, and semi-quantify their copper methanol:water (30:38:2) for sugar analysis. The sheets were uptake level. A few promising isolates with higher copper then air dried, sprayed with ninhydrin reagent (0.2% resistance ability were also characterized at the molecular ninhydrin in acetone) and acid aniline phthalate for DAP level with respect to 16S rRNA phylogenetic analysis as well and sugar, respectively, and heated at 100°C for 3 min. as plasmid isolation, pre- and post-electro-transformation Chromatographic fingerprints were compared with standards. perturbance. Plant growth attributes such as siderophore production were assayed according to Schwyn and Neilands (1987)by observing clear halo zone formation on Chrome Azurol S Materials and methods medium. For nitrate reduction, freshly grown cultures were inoculated in 10 mL peptone water and incubated for 48– Study site and soil characterization 72 h at 28°C. Nessler’s reagent (0.5 mL) was added to each tube and development of brown to yellow color was Sampling sites for present study were distributed across and considered as a positive test for ammonia production the adjoining districts of Gorakhpur, Mau, Gazipur, and (Cappuccino and Sherman 1992). Varanasi (23°52′ to 30°25′N, 77°30′ to 84°39′E) in Uttar Antimicrobial activity was evaluated on ISP-2 medium Pradesh, India. Soil samples were collected at 10- to 12-day using the streaking method. The isolate was inoculated in a interval from each site, in triplicate. Monoliths of 5 cm3 straight line on plate (90 mm Ø) and incubated at 28±2°C for were randomly removed between the chickpea rows, at least 14 days. Target microorganisms were seeded in cross aseptically stored in sterile polythene bags, brought to the streaks to Streptomycetes cultures. The antimicrobial activity laboratory and dried at 30°C to obtain constant weight. was evaluated by measuring the distance (mm) of inhibition Similarly, sampling was performed from chickpea rows to between target microorganisms and Streptomycetes colony obtain rhizospheric samples. Each composite soil sample margins. The challenged microorganisms were Bacillus was divided into two equal parts. One part in the field moist subtilis, Rhizoctonia solani and Macrophomina phaseolina. condition was used for determination of chemical and Hydrogen cyanide production (Lorck 1948) was assayed by physiological parameters, while other part was used for inoculating culture in nutrient broth amended with 4.4 g L−1 Streptomycetes isolation. Samples were kept at 4°C for of glycine, and Whatman No. 1 filter paper strip soaked in further analysis. Soil texture and physiochemical properties picric acid (0.05% solution) in 2% Na2CO3 (2 g in 100 mL) of soil samples were characterized according to standard was placed in the lid of each petriplate, sealed with paraffin, Ann Microbiol (2010) 60:605–614 607

Table 1 Soil characteristics of sampling sites Soil property Sampling cites

Varanasi Mau Gazipur Gorakhpur

R NRRNRR NRRNR

Texture Loam Chalky Silt Loam Chalky Chalky Loam Silt pH 8.1 8.9 8.7 8.4 8.9 9.2 8.1 9.5 E.C. 4.5 4.53 15.1 16.7 9.02 10.12 5.6 12.45 N (Kg ha−1) 256.41 238.64 144.32 148.02 288.64 200.48 132.86 152.03 P(Kg ha−1) 18.2 10.3 8.09 9.93 21.35 14.93 15.48 12.35 K (Kg ha−1) 198.04 158.56 176.43 102.25 147.28 92.26 192.78 164.90 R Rhizospheric soil, NR non- Na (Abs. ratio) 16 18 28 26 87 67 30 19 rhizospheric soil incubated at 28°C for 6 days and the results noted according (Mergeay 1995; Yilmaz 2003) of selected strains were to changes of the filter paper from deep yellow to orange. determined by saturating 6-mm-diameter discs with metal Physiological and biochemical characteristics such as opti- solutions of different concentrations, i.e., 0.075, 0.15, 0.22, mum temperature and pH for growth, carbon utilization 0.26, 0.50, 0.75, 0.94, 1.00, 1.25, 3.12, 4.37 and 5.03 mM pattern, and degradation assay (xanthine, casein, tyrosine), CuSO4. The discs were placed on the surfaces of Petri dishes were tested according to Williams et al. (1989). containing media previously inoculated with spores of the strain to be tested. The diameter of the growth inhibition was Scanning electron microscopy measured after incubation at room temperature for 7 days. The MIC were expressed in mMoles. Spore surface ornamentation of the isolates S160, 161 and 164 was observed by scanning electron microscopy Analysis of metal removal and growth kinetics studies analysis. Mycelia were taken (from 10-day-old plates) and washed in 0.1 M sodium cacodylate buffer (pH 7.4). They Semi quantative assay of isolates was determined by using were fixed in 2.5% glutaraldehyde in 0.1 M sodium MM medium (25 mL, 28±2°C, 200g) amended with different cacodylate buffer for 4 h at 4°C followed by post-fixation concentrations (0.075–5.03 mM, as above) of CuSO4 for with 1% OsO4 in 0.1 M sodium cacodylate buffer (pH 7.4) 7 days. Total microbial biomass was harvested by centrifu- and dried in a critical point dryer (EMITECH model K850; gation (12,000g, 15 min) in a Sigma 3 K30 centrifuge, Hitachi). The preparations were mounted onto aluminium washed twice with sterile deionized water, drained and dried holders, sputter-coated with 10 nm Au and observed by at 60°C until constant weight. Further, the pellets were SEM (Hitachi model S3400 at 15–30KV, 2–5.00 μm). treated with acid digestion (nitric acid:perchloric acid, 5:3), metal concentration in both washed fluid and pellets were Distribution pattern of isolates and minimal inhibitory determined using an atomic absorption spectrophotometer concentrations for copper metal (Brunswick, 301). All the metal removal experiments were conducted in triplicate and mean values were used for data For enumeration of spatial Streptomycetes populations, analysis. Parallel controls without inoculums and copper was monoliths were frozen at −20°C for 2 h, then carefully cut also analyzed. The spore suspension (1×109 CFU) used for from outside with a knife to create slices 5 mm thick, starting inoculation of rhizospheric and non-rhizospheric isolates was from outside and moving inwards. These slices were also prepared as described by Kieser et al. (2000). For growth processed as composite soil samples. The average distributions kinetics studies, resistant strains (S160, S161 and S164) were (CFU×102 g soil−1) of Streptomycetes from rhizospheric and inoculated in MM and incubated at 28±2°C (agitation rate, non-rhizospheric soils were counted by spread plate techni- 200g). Different concentrations of CuSO4 (0, 0.5, 0.75 and ques on Starch Casein Nitrate agar medium. 1.0 mM) were added after 6 h of incubation. Growth kinetics Primary qualitative screening was carried out according to was measured spectrophotometrically by monitoring absor- Amoroso et al. (1998) in minimal agar medium. Troughs bance at 600 nm at regular intervals. were made in the center of the plate and filled with 500 μL of CuSO4 solutions of different concentrations, i.e., 0, 0.075, Antibiotic resistance 0.15, 0.22, 0.26, 0.50, 0.75, 0.94, 1.00 and 1.25 mM. Microbial growth was used as the qualitative parameter of Antibiotic resistance was tested, using Nutrient agar metal resistance. Minimum inhibitory concentrations (MIC) containing vancomycin (30 μgmL−1), tetracycline 608 Ann Microbiol (2010) 60:605–614

Fig. 1 Average distribution pattern of Streptomyces population (CFU×102) from rhizospheric and non-rhizospheric soils samples from different regions of chickpea field

(20 μgmL−1), kanamycin (100 μgmL−1), rifampicin following profile: initial denaturation of 94°C for 30 s, (100 μgmL−1) and chloromphenicol (40 μgmL−1), which followed by 30 cycles with 94°C for 1 min, 52°C for 1 min, were added aseptically to the medium after autoclaving. and 72°C for 1.5 min, followed by 15 min final extension step at 72°C. PCR product were purified using PCR Molecular characterization and electrotransformation purification kit (Genei), and sequenced using the same primer studies set as used in PCR amplification on an ABI automatic sequencer (ABI Prism 3130XL). G-DNA was extracted by the modified method of Boudjella et Phylogenetic characterization and their identity were al. (2006) and its purity was checked by the ratio OD260/ determined by BLASTn result, sequences were aligned by OD280. PCR amplification of 16S rDNA isolates was done by Clustal-X, and the phylogenetic tree was constructed by Mega using universal primers fD1 (5′-GGTGGCGAAGGCGGA- 3.1 software package using neighbor-joining methods (Saitou 3′)andRP2(5′-GAACTGAGACCGGCTTTTTGA-3′) and Nei 1987). Bootstrap analysis was conducted using (Monciardini et al. 2002). PCR mixture contained 1.5 mM 1,000 resamplings of data.

MgCl2, 200 mM dNTP, 1 mM each primer, 5U of Taq Plasmid was extracted from the log phase cultures by polymerase (Genei, India) and 50 ng DNA template. lysing with 600 μL of lysis buffer (10 mM Tris-HCl, pH Amplification was done by Bio-Rad thermal cycler with the 7.5; 1 mM EDTA; 0.1 N NaOH; 0.5% w/v SDS) and

Fig. 2 Copper accumulated, loosely bound and total sorption profile of S160, S161 and S164 Streptomycetes strains. Bars = S160,

= S161 and = S164 Ann Microbiol (2010) 60:605–614 609

Fig. 3 Represents growth characteristics of copper resistant Streptomyces sp. at the concentration of a 0.5 mM, b 0.75 mM, and c 1.0 mM CuSO4 isolated from chickpea field. Symbols █ = S160, = S161 and = S164 precipitating with 320 μL of 3.0 M sodium acetate (pH was found to be greater in non-rhizospheric soils (51×102 5.2). The supernatant was extracted with phenol:chloro- to 96×102 CFU g soil−1) than in rhizospheric soils (31×102 form:isoamyl alcohol followed by 400 μL chloroform and to 64×102 CFU g soil−1 rhizospheric soil). The population precipitated with isopropanol and washed twice with 70% in the root zone was found to be lower in rhizospheric soil ethanol. The DNA pellet was resuspended in 40-60 μLof and the highest population was present at a distance of 0– Tris-HCl buffer (10 mM Tris-Cl, pH 8.5) containing RNase 5 mm from the root zone of chickpea and decreased H (20 μgmL−1), and finally plasmids were precipitated gradually moving towards the root zone in non- with absolute ethanol and resuspended in TE buffer and rhizospheric soil. The average proportion remained con- bands were documented (Sambrook and Russell 2001). stant regardless of sampling time during the cropping Electro-transformation was carried out according to the season (Fig. 1). procedure given by Tyurin et al. (1998) using susceptible Primary screening of rhizospheric and non-rhizospheric wild strains. isolates for Cu2+ resistance showed that 100, 62, and 8%

rhizospheric isolates could tolerate CuSO4 at the concentrations of 0.075, 0.15 and 0.22 mM, whereas non- Results rhizospheric isolates showed 0.075, 0.264 and 0.628 mM

of CuSO4, respectively. Minimum inhibitory concentration Distribution and primary screening of minimum inhibitory (MIC) of rhizospheric and non-rhizospheric isolates was concentrations of copper-resistant isolates recorded at the concentration of 0.264 and 0.94 mM

CuSO4, respectively, with a few exceptions, as one A total of 432 isolates (261 rhizospheric and 171 non- rhizospheric isolate, S160, showed MIC of 3.14 mM rhizospheric) were isolated from both rhizospheric and non- CuSO4, while two non-rhizospheric isolates, S164 and 2 rhizospheric soil samples by using spread plate (10 S161, showed MIC of 1.88 and 3.14 mM CuSO4, dilution) technique. Average density of Streptomycetes respectively (Fig. 2).

Fig. 4 Scanning electron micrographs (a, b, and c) of strains S160, S161, and S164, respectively 610 Ann Microbiol (2010) 60:605–614

Table 2 Physiological and biochemical characteristics of Characteristics S 160 S 161 S 164 the Streptomyces strains, S160, S161, and S164 Spore chain morphology Rf Rf Grape-like clusters Colony color on ISP-2 White White Brown Diffusible pigment Colorless Yellow Black Growth conditions Temperature 28±2°C 28±2°C 28±2°C pH 7.0 7.0 7.0 Degradation Xanthine + + + Tyrosine + + + Casein + + + Tween + - -

H2S production + + - Gelatin liquification - - + Urea - + + Nitrite reduction + + + Siderophore production - + - Rf Rectiflexible type of spore HCN production + - + chain morphology

Analysis of metal removal and growth kinetics were calculated for S160 (at 1.2, 6.0 and 14.4 h), S161 (at 7.2, 12.0 and 24.0 h), and S164 (at 4.8, 7.2 and 16.8 h), The time course of microbial growth of highest metal- respectively (Fig. 3). tolerant strain (S160) in the presence of 0.264 mM CuSO4 showed a growth inhibition of 10% after 72 h of cultivation Antibiotic resistance and 32% after 144 h. Residual copper estimation indicated a reduction of 68% of growth inhibition after 144 h of Cu2+-tolerant isolates (S160, S161 and S164) were incubation. It is important to note that during the first 72 h examined for antibiotic resistance and it was found that the initial copper concentration in the medium did not they are resistant to vancomycin (30 μgmL−1), tetracy- change appreciably. In contrast, between 72 and 120 h, cline (20 μgmL−1), kanamycin (100 μgmL−1)and there was a drastic decrease of residual copper (0.04 mM). rifamycin (100 μgmL−1), and chloromphenicol After 168 h of growth, 0.12 mM accumulated copper was (40 μgmL−1). After electro-transformation of resistant detected in acid-digested biomass. These results evidently species plasmids to wild-type susceptible strains, results indicate copper sorption ability of S160 strain. Microbial showed that acceptor wild strain showed resistance only to growth at different concentrations of Cu (0.5, 0.75 and rifampicin while negative resistance was expressed to 1.0 mM) was calculated by using a semi-logarithmic scale other antibiotics. and it was found that it enters into the stationary phase after 34, 43 and 38 h, respectively. Growth kinetics showed Electrotransformation studies and copper resistance optical density and maximum specific growth rate for capacity resistant strains (S160, S161 and S164) decreased with increasing concentrations of CuSO4, while generation time To examine the relationship between copper resistance and at different Cu2+ concentrations (0.5, 0.75 and 1.0 mM) presence of plasmids, plasmid-free wild-type strains isolated

Table 3 Antimicrobial activity of Streptomyces strains, S160, Microbial isolates Actinomycetes isolates (inhibition zone in mm) S161, and S164 S160 S161 S164

Gram-positive bacterium Bacillus subtilis 30±1.4 35±2.2 22±3.1 Fungal isolates Rhizoctonia solani - 20±2.3 22±1.4 Values represent average of five Macrophomina phaseolina 15±3.0 - - replications ± standard deviation Ann Microbiol (2010) 60:605–614 611

Table 4 Assimilation of different carbon sources of Streptomyces Strain characterization strains S160, S161 and S164

S. no. Carbon sources S160 S161 S164 The microscopic observations ascertained that spores of all the three Streptomycetes isolates were positive for Gram- 1 Adonitol - - - staining with spirals, loops and grape-like bunch forms 2 Cellobiose - - - (Fig. 4), having characteristic white aerial mycelium, 3 Citrate + + + degradation ability and PGP traits (Table 2), type I cell 4 D- Arabinoside - - - wall (LL-DAP and without characteristics sugars) indicating 5 Dextrose + + + that they belonged to the Streptomyces genus. They 6 Dulicitol - - - exhibited antimicrobial activity against (Gram-positive) 7 Esculin + + + Bacillus subtilis and Macrophomina phaseolina (Table 3). 8 Fructose + + - The detailed carbon utilization spectrum has been tabulated 9 Galactose - + + in Table 4. 10 Glucosamine - - - 11 Glycerol - - - Molecular characterization and phylogenetic analysis 12 Inositol - + - 13 Inulin - - + On the basis of BLASTn homology search of NCBI for 16S 14 Lactose - - _ rRNA gene sequences (600 and 468 bp approx.) (Fig. 5a), 15 L-Arabinose - - - all the three strains were confirmed to be Streptomyces sp., 16 Malonate + + + whereas plasmids isolated from S160, S161, and S164 were 17 Maltose + + + designated as pS160, pS161, and pS164 and their estimated 18 Mannitol + + - sizes were approximately 3.1 kb, 2.8 kb, and 2.5 kb, 19 Mannose - - - respectively (Fig. 5b). 20 Melazitose - - + The phylogenetic analysis showed mainly three groups 21 Melibiose - - - i.e., strains S160 and S161 showed 100% similarity with 22 Ortho-nitro phenyl-β-D- +- - Streptomyces sp.; however, the strain S164 had 99% galactopyranoside (ONPG) similarity with Streptomyces hygroscopicus. The gene 23 Raffinose - - - sequences of S160, S161 and S164 were submitted to 24 Rhamnose - - - GenBank(NCBI)withaccessionnumbersGQ884178, 25 Ribose - + + GQ884179 and GQ884180, respectively (Fig. 6). 26 Salicin - - - 27 Sodium gluconate - - - 28 Sorbitol + - - Discussion 29 Sorbose - - - 30 Sucrose + - + Cosmopolitan Streptomycetes have wide variations in their 31 Trehalose - + + ecological and population dynamics. By geochemical 32 Xylitol - - - processes, living organisms have been exposed to heavy 33 Xylose - - - metals in the environment (Brown et al. 1998). It is not 34 α methyl- d-glucoside - + - surprising to find a prevalence of copper resistance ability 35 α methyl-d- mannoside + - - among actinomycetes. Moreover, isolates from rhizospheric soils of chickpea in the present work have been demon- + Assimilation, - non-assimilation strated to be more resistant than strains from non- rhizospheric soil, which may firmly indicate and postulate the presence of induction mechanisms towards copper resistance. The possibility of inducibility was checked by in this study were transformed (≥92% efficiency) with conjugating resistant isolates with non-resistant ones. The plasmids of resistant strains, S160, S161 and S164. All results strongly postulate that resistance-imparting factors susceptible isolates acquired similar resistant ability to copper are plasmid encoded, partially if not fully. In addition, after electro-transformation. Metal resistance ability along antibiotic-resistance profiles were also acquired by recipi- with rifamycin antibiotic resistance capacity was acquired ents further indicating that metal resistance and antibiotic similar to the donor strains. This indicated the location of resistance encoded genes are also clustered together on heavy metal resistance genes and rifampicin resistance genes plasmids. According to Jeffery’s Selfish operon theory, on plasmid DNA of donor strains. clustering of genes on the plasmid create a beneficial 612 Ann Microbiol (2010) 60:605–614

resistance locus in these microorganisms. Similar results were also obtained by Abbas and Edwards (1990) for S. coelicolor with a growth inhibition of 50% after 6 days of incubation in starch–yeast extract broth supplemented with 3mgL−1 of Cu2+. Amoroso et al. (1998) showed that the actinomycete strain R25 had an inhibition of 40% after 48 h of growth in a minimal liquid medium amended with −1 32 mg L of CuSO4. It is important to note that the use of minimal medium for the growth of actinomycetes assures us that the supplemented metal does not form complexes with components of the medium, and that all the metal is available for biosorption (Amoroso et al. 1998). Previous studies used complex media with higher concentrations of copper where sequestration of the metal is expected, for instance especially with amino acids like cysteine, methi- onine or hystidine that are proven to bind copper (Koch et al. 1997). Isolate S160 showed the ability to remove more than 65% of copper from the culture medium (0.47 mM) in only 72 h, indicating that it is a suitable agent for bioremediation of soils or effluents with high concentrations of copper. This copper removal and retention ability observed in the S160 strain may involve a fully integrated system of uptake, storage and distribution of the metal, present in Streptomyces strains. In the present study, S160, S161 and S164 strains have been identified as copper Fig. 5 a 16S rDNA amplified product of Streptomyces strains S160, resistant up to the level of 5.03, 3.14 and 1.88 mM, and S161 and S164 (Lane M 1 kb DNA ladder, Genei, India; Lane 1 S160; their growth characteristics reported herein match the work Lane 2 S161; Lane 3 S164). b Plasmid profile of Streptomyces strains reported earlier by Mahapatra and Banerjee (1996). All the S160, S161 and S164. (Lane 1 Shigella flexneri 49 used as size strains reported in this study utilized various carbon standard; Lanes 2 and 6 Control; Lanes 3, 4 and 5 the size of plasmids pS160, pS161 and pS164 from strains S160, S161 and S164 of sources, as utilization of a variety of carbon sources helps approximately 3.1, 2.8 and 2.1 kb, respectively) in adaptation to a variety of inoculation sites and wide soil types. Actinomycetes–microbial antagonism has been condition for the survival of the organism and its species. reported for a wide variety of fungal, bacterial and Thus, in an environment with multiple stresses, antibiotic oomycetes plant pathogens like Rhizoctonia (Rothrock and heavy metal, it would be more ecologically favorable in and Gottlieb 1984), Phytophthora fragariae, Fusarium terms of survival. Further genetic manipulation will help in (Valois et al. 1996), Macrophomina (Hussain et al. 1990), understanding the physiological and genetic basis of copper Verticillium spp. (Wadi and Easton 1985), and B. subtilis

Fig. 6 Phylogenetic analysis of Streptomyces isolates from chickpea fields based on partial nucleotide sequences (0.6 kb approx) of the 16S rDNA. The tree was constructed using the neighbor-joining method. Percentages at nodes represent levels of bootstrap support from 1,000 resampled datasets. Bootstrap values less than 50% are not shown. Bar 0.001% estimated sequence divergence Ann Microbiol (2010) 60:605–614 613

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