Identification of Luteomycin like Antibiotic Produced by Streptomyces tanashiensis AZ-C442 Isolated from Luxor Governorate at Upper Egypt

INTRODUCTION

Luteomycin is similar to, Tautomycetin (TMC) is an unusual linear polyketide compound esterified with a cyclic anhydride. It exhibits novel activated T cell-specific immunosuppressant as well as anti-cancer activities. It was isolated and characterized the entire TMC biosynthetic gene cluster from Streptomyces sp. Since many antibiotics are able to suppress or retard the growth of tumors, an extensive effort has been made in many laboratories to find new antibiotics with antineoplastic properties either by screening new soil isolates or by chemical modification of the existing antibiotics such as our goal in this study. Because the Actinomycetes are the source of most clinically used antibiotics, as well as of several widely used drugs against common diseases, including cancer (Medema et al., 2011) and their genome sequencing revealed that the potential of Streptomyces species for the production of valuable secondary metabolites is high. It is well documented that the biosynthesis of Streptomyces secondary metabolites is typically regulated via multiple regulatory pathways operating with several layers of complicated control systems (Chen et al., 2010; Lee et al., 2005).

The comparison of rRNA sequences is a particularly powerful tool in streptomycete taxonomy. rRNA sequence comparisons have also been useful for answering questions concerning the horizontal transfer of genes within the genus Streptomyces (Huddleston et al., 1997). The nucleotide sequence of the 16S rRNA gene of Streptomyces sp. was determined (Edwards et al., 1989; Wang et al., 2011), the PCR product was analyzed by agarose gel electrophoresis and the DNA fragment with the expected size was purified and sequenced (Palaniappan et al., 2009; Wang et al., 2011).

Cultural criteria, such as growth on different media, the color of aerial and substrate mycelia and the formation of soluble pigments among others and ecological properties has also been used for the classification of Streptomyces sp. (Shirling and Gottlieb, 1966; Lechevalier and Lechevalier, 1980). Colour names were assigned to the mycelial and diffusible pigments on the basis of the Inter-Society Color Council-National Bureu of Standards (ISCC-NBS) centroid color charts (Burres et al., 1995). Many investigators have attempted to obtain optimal liquid culture requirements for obtaining a maximum yield of antibiotic production by different Streptomyces species (Yoshimoto et al., 2000; Momose et al., 2001; Kurosawa et al., 2001; Fang, 2002; Huang et al., 2003; Xu and Yun, 2003; Atta et al., 2011).

In the course of antibiotic purification, different solvents were used and tested for the extraction of the antibiotic, in case of S. albidoflavus PU 23, maximum antibiotic yield was observed in residue, which was extracted by using n-hexane and purified by column chromatography using silica gel (Augustine et al., 2005). On the same context, El-Naggar et al. (2006) analysed Meroparamycin antibiotic produced by newly isolated Streptomyces sp. by elemental analysis method such as IR (infrared spectrophotometer), UV absorption, high and low-resolution mass spectra and using the Electron Impact (EI) method to elucidate its structure.

Also, UV or MS detection are the most popular techniques in antibiotics analysis, however, fluorescence has also been used successfully (Diaz-Cruz et al., 2003). Moreover, the actinomycins are characterized using a variety of analytical methods including ultraviolet visible spectroscopy system, Infrared (IR), electrospray ionization mass spectrometry (ESI-MS) and tandem mass spectrometry (MS-MS) as well as various NMR techniques (Singh and Gurusiddaiah, 1984; Kurosawa et al., 2006). The absorption spectrum of active extracts in methanol were recorded in the UV region (210-400 nm) by using a UV-vis spectrophotometer (Cintra 40) and compared with those of known polyenic antifungal antibiotics (Thakur et al., 2007). Many authors have synthesized antibiotics which possess biological activities including, antimicrobial, antifungal, anti-tumor, antimalarial and antiparasitic effects (Laursen and Nielsen, 2004; Mavrodi et al., 2010, Wang et al., 2011; Atta et al., 2011).

This investigation aimed to isolate and identify the antimicrobial producing Sterptomycete sp. from the soil in Upper Egypt and to isolate, purify and identify the produced antimicrobial agent as well as evaluate its MIC.

MATERIALS AND METHODS

Microorganism: The actinomycete AZ-C442 was isolated from soil sample collected from Luxor governorate. It was purified using the soil dilution plate technique described by Williams and Davis (1965).

Taxonomic studies of actinomycete isolate (AZ-C442)
Morphological characteristics: Morphological characteristics of aerial hyphae, spore mass, spore surface, color of aerial and substrate mycelia and soluble pigments production were conducted by growing the organism on starch-nitrate agar medium and yeast extract-malt extract agar medium.

Physiological and biochemical characteristics: Lecithinase was detected using egg-yolk medium according to the method of Nitsch and Kutzner (1969), lipase (Elwan et al., 1977), protease (Chapman, 1952), pectinase (Hankin et al., 1971), α-amylase (Ammar et al., 1998) and catalase test (Jones, 1949). Melanin pigment (Pridham et al., 1957), esculin broth and xanthine have been done according to Gordon et al. (1974). Nitrate reduction was performed according to the method of Gordon (1966). Hydrogen sulphide production was carried out according to Cowan and Steel (1974). The utilization of different carbon and nitrogen sources was carried out according to Pridham and Gottlieb (1948).

Determination of Diaminopimelic acid (DAP) and sugar pattern were carried out according to Becker et al. (1964) and Lechevalier and Lechevalier (1970).

Color characteristics: The ISCC-NBS color-name charts illustrated with centroid detection of the aerial, substrate mycelia and soluble pigments (Kenneth and Deane, 1955) was used.

DNA isolation and manipulation: The locally isolated actinomycete strain was grown for 6 days on a starch agar slant at 30°C. Two milliliter of a spore suspension were inoculated into the starch-nitrate broth and incubated for 4 days on a shaker incubator at 200 rpm and 30°C to form a pellet of vegetative cells (pre-sporulation). The preparation of total genomic DNA was conducted as described by Fritsch et al. (1989).

PCR amplification and sequencing of the 16S rRNA gene: PCR amplification of the 16S rRNA gene of the local actinomycete strain was conducted using two primers, StrepF; 5.-ACGTGTGCAGCCCAAGACA-3. and Strep R; 5.ACAAGCCCTGGAAACGGGGT-3 (Edwards et al., 1989). The PCR mixture consisted of 30 pmol of each primer, 100 ng of chromosomal DNA, 200 μM dNTPs and 2.5 units of Taq polymerase, in 50 μL of polymerase buffer. Amplification was conducted for 30 cycles of 1 min at 94°C, 1 min of annealing at 53°C and 2 min of extension at 72°C. The PCR reaction mixture was then analyzed via agarose gel electrophoresis and the remaining mixture was purified using QIA quick PCR purification reagents (Qiagen, USA). The 16S rRNA gene was sequenced on both strands via the dideoxy chain termination method (Sanger et al., 1977).

Sequence similarities and phylogenetic analysis: The BLAST program (Altschul et al., 1997) was employed in order to assess the degree of DNA similarity. Multiple sequence alignment and molecular phylogeny were evaluated using BioEdit software (Hall, 1999). The phylogenetic tree was displayed using the TREE VIEW program.

Parameters controlling antimicrobial agent biosynthesis: These parameters included, incubation period and temperature, agitation and aeration, pH values, carbon source and nitrogen sources, vitamins, MgSO₄.7H₂O and K₂HPO₄ concentrations, inoculum age and size, amino acids and the medium kinds. All these parameters have been determined by the standard methods.

Purification of antimicrobial agent
Fermentation: Streptomyces tanashiensis, AZ-C442 inoculum was introduced aseptically into each sterile flasks containing the following ingredients (g L^-1): Glycerol, 20; NaNO₃, 2.0; K₂HPO₄, 0.6; MgSO₄.7H₂O, 0.5; Vitamin B₁₂, 200 (ppm) and KCl, 0.5. The pH was adjusted at 8.0 before sterilization. After 10 days of incubation at 35°C filtration was carried out through filter paper Whatman No. 1 and followed by centrifugation at 5000 rpm for 15 min. Only clear filtrates were tested for their antimicrobial activities (Eladly, 2009).

Extraction: The clear filtrates were adjusted at different pH values (4 to 9) and extraction process was carried out using different solvents separately at the level of 1:1 (v/v). The organic phase was concentrated to dryness under vacuum using a rotary evaporator.

Precipitation: The precipitation process of the crude compound was carried out using petroleum ether (bp 60-80°C) followed by centrifugation at 5000 rpm for 15 min. The precipitate was tested for its antimicrobial activities.

Separation: Separation of the antimicrobial compound into its individual components was conducted by thin layer chromatography using chloroform and methanol (24:1, v/v) as a solvent system.

Purification: The purification of the antimicrobial compound was carried out using silica gel column (2.5x50) chromatography. Chloroform and Methanol 10:2 (v/v) was used as an eluting solvent. The column was left overnight until the silica gel (Prolabo) was completely settled. One milliliter crude extract to be fractionated was added on the silica gel column surface and the extract was adsorbed on top of silica gel. Fifty fractions were collected (each of 5 mL) and tested for their antibacterial activities.

Physico-chemical properties of antimicrobial agent
Elemental analysis: The elemental analysis of C, H, O, N and S were carried out at the Microanalytical Center, Cairo University, Egypt.

Spectroscopic analysis: The IR, UV and Mass spectrum were determined at the Micro analytical Center, Cairo University, Egypt.

Biological activity: The Minimum Inhibitory Concentration (MIC) has been determined by the cup method assay (Kavanagh, 1972).

Characterization of the antimicrobial agent: The antibiotic produced by Streptomyces tanashiensis AZ-C442 was identified according to the recommended international references of Umezawa (1977) and Berdy (1974, 1980a-c).

RESULTS

Isolation, purification and bioactivity of actinomycete isolates: In a previous study, we did isolation and purification of actinomycete colonies (the broadest source of antibiotics) from 40 soil samples collected from various Egyptian localities and found that the highest number of isolates (84) out of 194 (43.2%) were isolated on starch nitrate agar (SNA) medium followed by 55 isolates (28.3%) on both starch casein agar and glycerol asparagine agar. The screening test for 194 actinomycete isolates, against certain bacteria, fungi and yeast, confirmed that the highest percentage (38%) 74 active isolates was obtained against Staphylococcus aureus 90.5% (67) followed by Aternaria alternata 43.2% (32) and klebsiella pneumoniae, 41.8% (31), while the lowest percentage was obtained against Fusarim verticillioides 21.6% (16 isolates), Salmonella typhi 20.2% (15 isolates), Escherichia coli 9.4% (7 isolates), Aspergillus fumigatus 9.4% (7 isolates), Saccharomyces cerevisiae 8.1% (6 isolates) and Aspergillus flavus 6.7% (5 isolates). Consequently, the previous above screening test indicated two Actinomycete isolates (AZ151 and AZ-C442) showed high potencies against all microorganisms tested. Thus, all items in this investigation are concerned with the most active Actinomecete AZ-C442, isolated from Luxor governorate, for production of antimicrobial agent.

Characterizations of the actinomycete isolate AZ-C442
Morphological characteristics: Spore chains were rectiflexibiles, spore masses were gray, spore surfaces were smooth and the reverse color pale yellow-light yellowish brown, while, diffusible pigment not produced (Fig. 1).

Cell wall hydrolysate: The cell wall hydrolysate contains LL-diaminopimelic acid (LL-DAP) and sugar pattern not detected (Table 1).

Physiological and biochemical characteristics: The actinomycete isolate AZ-C442 could hydrolyze starch, protein, lipid and pectin, whereas catalase was negative. Melanin pigment production, degradation of xanthin, production of H₂S, decomposition of urea, nitrate reduction, utilization of citrate and KCN were positive but esculin decomposition is negative.

The isolate under study utilizes D-xylose, D-mannose, D-glucose, D-galactose, L-arabinose and trehalose but do not utilize sucrose, rhamnose, raffinose, mannitol, meso-inositol, lactose, maltose, D-fructose and ribose.

Fig. 1:	Scanning electron micrograph of the actinomycete isolate AZ-C442 growing on starch nitrate agar medium showing spiral and rectiflexibiles spore chains and had a smooth surface. Neither sclerotic granules, sporangia nor flagellated spores were observed (X15, 000)

Table 1:	The morphological, physiological and biochemical characteristics of the actinomycete isolate AZ151
-: Negative, +: Positive, ±: Doubtful results and ++: Moderate growth

It had good growth on L-glycine, L-histidine, L-asparagine, L-phenyl alanine and L-lysine and no growth on L-valine, L-leucine and L-methionine. Growth was inhibited in the presence of, up to 3% NaCl and of 0.01% (w/v) sodium azide but the growth was not inhibited in the presence of 0.1% (w/v) phenol and at 45°C. The actinomycete isolate was resistant to nalidixic acid (30 μg mL^-1), cefoperazone (75 μg mL^-1) and fusidic acid (10 μg mL^-1), whereas not resistant to ampicillin (25 μg mL^-1), polymyxin (30 μg mL^-1), gentamicin (10 μg mL^-1) and kanamycin (30 μg mL^-1) (Table 1).

Table 2:	Culture characteristics of the actinomycete isolate AZ-C442
*The color of the organism under investigation was consulted with the ISCC-NBS color-name charts illustrated with centroid color

Color and culture characteristics: Data recorded in Table 2, declared that the growth on AZ-C442 strain was disappeared on ISP-1, moderate on ISP-2, 3 and 5 and good on SNA and ISP- 4, 6 and 7. While, the aerial mycelium was light gray on almost all media used except ISP-1, substrate mycelium has been appeared, light gray yellowish brown on SNA and ISP-7, light yellowish brown on ISP-2, 3 and 6 and pall orange yellow on ISP- 4 and 5. Culture characteristics was not detected on ISP-1. Diffusible pigments were only detected on SNA and ISP- 6 and 7 (Table 2).

Identification of actinomycete isolate, AZ-C442: This was performed basically according to the recommended international Key’s viz. (Buchanan and Gibsons, 1974; Holt and Williams, 1989; Hensyl, 1994). On the basis of above collected data and in view of the comparative study of the recorded properties of AZ-C442 in relation to the most closest reference strain, viz. Streptomyces tanashiensis (CHR and C442) (Table 3), it could be concluded that both isolates are identical on the basis of spore mass is gray, spore chain is retiflexibilies and spores are non motile. Cell wall hydrolysate contains LL-diaminopimelic acid and sugar pattern not detected. Melanin pigment not produced. They utilize D-xylose, D-glucose, D-galactose and L-arabinose but do not utilize sucrose, rhamnose, raffinose, mannitol, meso-inositol and D-fructose.

In view of all the previous characteristics of AZ-C442, it could be stated that it is suggestive of being belonging to Streptomyces tanashiensis and thus given the name Streptomyces tanashiensis AZ-C442.

Molecular phylogeny of the selected isolate: The 16S rDNA sequence of the local isolate was determined as 721 bp as shown in Table 4 and compared to the sequences of Streptomyces spp. in order to determine the relatedness of the local isolate to these Streptomyces strains. The phylogenetic tree (as displayed by the Tree View program) revealed that the locally isolated strain is closely related to Streptomyces sp., the most potent strain evidenced an 89% similarity with Streptomyces tanashiensis (Fig. 2).

Table 3:	A comparative study of the characteristic properties of AZ-C442 in relation to reference strain, Streptomyces tanashiensis. (Bergey’s 1989)
-: Negative and +: Positive results

Table 4:	Multiple sequence alignment was conducted the sequences of the 16S rDNA gene of Streptomyces tanashiensis

Parameters controlling antimicrobial agent biosynthesis: The evaluation of different environmental and nutritional factors on the biosynthesized antimicrobial activity indicated that the maximum activity was exhibited in starch nitrate broth medium at, incubation period (8 days), incubation temperature (30°C), agitation and aeration (120 rpm), pH value (8.0), carbon source (glycerol), nitrogen source (NaNO₃), water soluble vitamin (Vitamin B12), inoculum age (15 days), inoculum size (10 %,v/v), amino acid (L-Asparatic acid), MgSO₄.7H₂O concentration (0.5 g L^-1, w/v) and at K₂HPO₄ concentration (0.08 %,w/v) (Table 5).

Fig. 2:	The phylogenetic position of the local Streptomyces sp. strain among neighboring species. The phylogenetic tree was based on the pairwise comparisons of 16S rDNA sequences

Fig. 3:	Fractionation pattern of antimicrobial agent produced by Streptomyces tanashiensis AZ-C442

Fermentation and isolation of antimicrobial agent: The fermentation process was carried out, using the above parameters, of liquid starch nitrate medium as production medium. Filtration was conducted followed by centrifugation at 4000 rpm for 15 min. The clear filtrates containing the active metabolite, was adjusted to pH 8.0 then extraction process was carried out using Ethyl acetate at the level of 1:1 (v/v). The organic phase was collected and evaporated under reduced pressure using rotary evaporator. The residual material was dissolved in least amount of DMSO and filtered. The filtrates were tested for their antibacterial activities.

The antimicrobial agent was precipitated by petroleum ether (bp 60-80°C) and centrifuged at 4000 rpm for 15 min. The fraction was test for antimicrobial activities. Separation of antibacterial agent into individual components was carried out by thin-layer chromatography using a solvent system composed of chloroform and methanol (24:1, v/v). Among three bands developed, only one band at R_f 0.75 showed antibacterial activity. The purification process through column chromatography packed with silica gel indicated that the most active fractions against the tested organisms ranged between 19 and 28 (Fig. 3)

Physicochemical characteristics of the antimicrobial agent: The purified antimicrobial agent produced by Streptomyces tanashiensis AZ-C442 has a characteristic odour, its melting points is 190°C. The compound is freely soluble in chloroform, ethyl acetate, n-butanol, acetone, ethyl alcohol, methanol and 10% isopropyl alcohol but insoluble in petroleum ether, hexane and benzene.

Table 5:	The environmental conditions and nutritional requirements affecting the biosynthesis of antimicrobial agent by Streptomyces tanashiensis AZ-C442
*Mean values of triplicate determinations were calculated. **SN: Starch nitrate medium, SC: Starch casein medium , YEME: Yeast extract malt extract medium, GA: Glycerol asparagine medium, ISS: Inorganic salt starch medium, TYE: Tryptone yeast extract medium

Fig. 4:	Infrared spectrum of the antimicrobial agent

Fig. 5:	Ultraviolet absorbance of antimicrobial agent

Elemental analysis: The elemental analytical data of the antibacterial agent revealed the following data: C = 59.61, H = 6.32, N = 2.97, O = 31.1 and S = 0. 0. This analysis indicates a suggested calculated empirical formula of C₂₆H₃₃NO₁₂.

Spectroscopic characteristics: The spectroscopic analysis of the purified of antimicrobial agent, produced by Streptomyces tanashiensis AZ-C442 indicated that, the Infrared (IR) spectrum of antimicrobial agent shows a characteristic band corresponding to 19 peaks (Fig. 4), the Ultraviolet (UV) spectrum record a maximum absorption peak at 280 and 420 nm (Fig. 5) and the Mass spectrum showed that the molecular weight is 552.52 (Fig. 6).

Biological activities of the antimicrobial agent: Data recorded in Table 6 indicated that the antimicrobial agent is fairly active against both Gram positive and Gram negative bacteria and unicellular and filamentous fungi.

Fig. 6:	Mass spectrum of antimicrobial agent.

Table 6:	Mean diameters of inhibition zones (mm) caused by 100 μL of the antimicrobial activities produced by Streptomyces tanashiensis AZ-C442 in the agar plate diffusion assay (The diameter of the used cup assay was 10 mm)

The Minimum Inhibitory Concentration (MIC) of antibiotic produced by Streptomyces tanashiensis AZ-C442 was determined and results showed that the MIC of antibiotic (μg mL^-1) against S. aureus (2.6), K. pneumonia (7.8), E. coli and S. typhi (15.6), A. flavus and A. alternata (46.87) and S. cerevisiae (62.5).

Identification of the antimicrobial agent: On the basis of the recommended keys for the identification of antibiotics and in view of the comparative study of the recorded properties of the antimicrobial agents, it could be stated that the antimicrobial agent is suggestive of being belonging to Luteomycin antibiotic (Table 7).