Ann Microbiol (2012) 62:1049–1057 DOI 10.1007/s13213-011-0346-y

ORIGINAL ARTICLE

A novel strain of Actinopolyspora mortivallis with antibacterial activity isolated from a Saharan soil

Atika Meklat & Nasserdine Sabaou & Noureddine Bouras & Abdelghani Zitouni & Cathrin Spröer & Hans-Peter Klenk & Florence Mathieu & Ahmed Lebrihi

Received: 21 March 2011 /Accepted: 29 August 2011 /Published online: 17 September 2011 # Springer-Verlag and the University of Milan 2011

Abstract A new halophilic actinomycete strain, designated hybridization confirmed that it belonged to A. mortivallis. H16, has been isolated from a hypersaline Saharan soil in This strain showed high activity against Klebsiella Ouargla province (southern Algeria) and characterized pneumoniae and optimally secreted antibiotics during the taxonomically using a polyphasic approach. The strain mid-stationary growth phase in liquid culture. The anti- grew at 18–50°C, pH 5–9, and 7–32% (w/v) NaCl. It biotics were extracted with n-butanol and separated on produced abundant aerial mycelia, which formed long silica gel plates by ethanol–ammonia–water (mobile chains of rod-shaped spores at maturity, and fragmented phase). The results of bioautography revealed the presence substrate mycelia. The strain contained chemotaxonomic of five antibiotics. The chemical revelations showed that markers that were diagnostic for the genus Actinopolyspora, these antibiotics were glycosylated polycyclic aromatic such as meso-diaminopimelic acid, arabinose, and galactose, compounds containing amine groups and hydroxamic phosphatidylcholine as a diagnostic phospholipid, and acids. The UV-visible and mass spectra of the most active predominant menaquinones MK-9(H4) and MK-10(H4). antibiotics were determined. The predominant fatty acids were anteiso- and iso-C17:0, iso-C15:0, and 9-methyl-C16:0. Phylogenetic analysis, based Keywords Halophilic actinomycete . Actinopolyspora on 16S rDNA gene sequences, confirmed that strain H16 is a mortivallis . Saharan soil . Taxonomy. Antibiotic activities . member of the genus Actinopolyspora and most closely Microbial natural products related to A. mortivallis (98.7% identity). DNA–DNA

Introduction A. Meklat : N. Sabaou (*) : N. Bouras : A. Zitouni Laboratoire de Recherche sur les Produits Bioactifs et la Valorisation de la Biomasse, Ecole Normale Supérieure de Kouba, Actinomycetes are a group of microorganisms that are BP 92, 16 050 Vieux-Kouba, Algiers, Algeria widely distributed in nature. Several studies on the e-mail: [email protected] ecology of actinomycetes have revealed that these microorganisms may occur in unusual habitats, such as C. Spröer : H.-P. Klenk German Collection of Microorganisms saline aquatic and terrestrial environments (Okazaki and and Cell Cultures (DSMZ), Okami 1975). The ecological distribution of actinomy- Inhoffenstraße 7B, 38124 Braunschweig, Germany cetes from Saharan soils has been studied in Algeria and their biodiversity in those soils demonstrated (Sabaou et F. Mathieu : A. Lebrihi Laboratoire de Génie Chimique UMR 5503 (CNRS/INPT/UPS), al. 1992, 1998). ENSAT/INP de Toulouse, Université de Toulouse, Antibiotics are produced by microorganisms, and actino- 1 avenue de l’Agrobiopôle, Castanet–Tolosan Cedex, France mycetes are likely the most important group of antibiotic- producing organisms (Demain 2006). This group, in A. Lebrihi Université Moulay Ismail, particular members of the genus Streptomyces, has been Marjane 2, BP 298 Meknes, Maroc studied in great depth, which has led to the discovery of a 1050 Ann Microbiol (2012) 62:1049–1057 large number of novel antibiotics (Okami and Hotta 1988). Cultural and micro-morphological characteristics In recent years, the rate of discovery of new antibiotics in the genus Streptomyces has been declining, and the number Cultural characteristics were determined after 1, 2, and 3 of drug-resistant pathogens have increased. The isolation of weeks of incubation on ISP-2, ISP-4 (Shirling and Gottlieb yet-unknown actinomycetes producing antibiotics appears to 1966), CMA, and nutrient agar media. The colors of the be essential in order obtain alternative antipathogenic agents substrate and aerial mycelia and any soluble pigments for those antibiotics that are becoming increasingly ineffective produced were determined by comparison with chips from and to find novel commercially valuable antibiotics. Several ISCC-NBS color charts (Kelly and Judd 1976). Spores and members of the families Micromonosporaceae (Actinoplanes, mycelia were examined by light microscopy (B1 series; Micromonospora) and Pseudonocardiaceae (Amycolatopsis, Motic, Xiamen, China) and scanning electron microscopy Saccharopolyspora) are considered among the major producers (model S450; Hitachi, Tokyo, Japan) after 3 weeks of of commercially important biomolecules (Solanki et al. growth on ISP-2 medium. 2008). The selective isolation and characterization of rare taxa from unexplored habitats could represent a supplementary Chemotaxonomic characterization source of novel bioactive molecules. In this context, the saline soils of south Algeria make up a large part of the Sahara and For the chemotaxonomic analyses, strain H16 was grown in may be of interest as a source of such bioactive molecules. complex medium broth (CMB) containing 15% (w/v) NaCl A broad range of biologically active molecules are at 30°C for 10 days on a rotary shaker (250 rpm). Biomass synthesized through pathways mediated by the polyketide was harvested by centrifugation at 3,500 rpm and washed synthetases (PKS)- and nonribosomal peptide synthetases several times with distilled water. Analysis of diaminopimelic (NRPS). The genes for these enzymes have been detected in acid and whole-cell sugars was carried out using the methods many actinomycetes producing antibiotics (Ayuso-Sacido and of Becker et al. (1964) and Lechevalier and Lechevalier Genilloud 2005;Metsä-Keteläetal.1999). Recent explora- (1970). Phospholipids were analyzed according to the tions of microbial antibiotic potential have focused on the procedures developed by Minnikin et al. (1977). The cellular screening of PKS and NRPS genes (Gontang et al. 2010; fatty acid composition was studied as described by Sasser Janso and Carter 2010; Qin et al. 2009). (1990) using the microbial identification system (MIDI). Here we describe the isolation of a new Actinopolyspora strain, designated H16, from a hypersaline Saharan soil Physiological characterization sample and its identification by conventional and molecular methods, together with the production and the partial Seventy physiological tests were used to characterize characterization of the corresponding antibiotics. actinomycete strains. Production of melanoid pigments was tested on ISP-6 and ISP-7 media (Shirling and Gottlieb 1966). Degradation of adenine, gelatin, guanine, hypoxanthine, Materials and methods milk casein, starch, testosterone, Tween 80, tyrosine, and xanthine was studied as described by Goodfellow (1971)and Actinomycete strain Marchal et al. (1987). Utilization of 23 carbohydrates and decarboxylation of nine organic acids were determined During an investigation of actinomycete diversity in according to the methods of Gordon et al. (1974). Lysozyme Saharan soils, strain H16 was isolated from a hypersaline sensitivity was evaluated by the method of Gordon and Barnett soil sample (electrical conductivity 33.9 mS cm–1) collected (1977). Growth at different temperatures (18, 25, 30, and from Ouargla province (southern Algeria), by plating 1:10 45°C), pH (5, 7, and 9), and NaCl concentrations (0, 7, 10, 15, serial dilutions of the sample on humic acid–vitamins agar 20, 25, 28, and 32% w/v) and in the presence of chloram- (Hayakawa and Nonomura 1987) supplemented with 20% phenicol (25 μgmL–1), erythromycin (10 μgmL–1), (w/v) NaCl at 30°C for 25 days. The choice of this medium kanamycin (5 μgmL–1), penicillin (25 μgmL–1), and was based on the good results obtained previously in our streptomycin (10 μgmL–1) was determined on nutrient agar laboratory for the isolation and selection of actinomycetes medium. All media used for physiological tests contained (Zitouni et al. 2005). The strain was purified and 15% (w/v) NaCl (except for the NaCl concentration test). maintained on complex medium agar (CMA) described by Chun et al. (2000) (casamino acids, 7 g; yeast extract, 10 g; DNA preparation, PCR amplification, sequencing, sodium citrate, 3 g; magnesium sulfate, 10 g; potassium and phylogenetic analysis chloride, 2 g; iron sulfate, 1 mL of a solution of 4.98% w/v; agar 20 g; distilled water, 1 L; pH 7.2) supplemented with Strain H16 was grown in 100 mL of the CMB supple- 20% (w/v) NaCl at 4°C. mented with 15% (w/v) NaCl. Biomass was harvested by Ann Microbiol (2012) 62:1049–1057 1051 centrifugation (8,000 rpm for 10 min) and washed twice with and Klebsiella pneumoniae E40), two filamentous fungi double-distilled water. Chromosomal DNA was prepared (Aspergillus niger CX33N and Penicillium expansum using a DNA extraction kit (JetFlex, Germany). The 16S B831P), and one yeast (Candida albicans M1). Strains rRNA gene was enzymatically amplified using the oligonu- of S. aureus and E. coli were from the collection of the cleotide primers 10-30 F (5′-GAGTTTGATC-CTGGCTCA- Pasteur Institute in Paris (France); those of A. baumanii, 3′) and 1500R (5′-AGAAAGGAGGTGATCCAGCC-3′), as E. cloacae,andK. pneumoniae were isolated from sick described by Rainey et al. (1996). Amplification was carried patients in hospitals of Algeria. The strains of filamentous out in a 50-μL reaction volume containing 1.5 U of fungi belonged to our collection and were producers of AmpliTaq Gold Taq polymerase (Applied Biosystems, Foster mycotoxins (ochratoxin A for A. niger and patulin and City, CA), dNTPs (0.25 mM each), 1 μMofeachprimer, citrinin for P. expansum). Actinomycetes were grown on and 100 ng of genomic DNA. Reaction conditions were: ISP-2 medium containing 15% (w/v) NaCl at 30°C for 97°C for 4 min, followed by 35 cycles of 97°C for 45 s, 12 days and then transferred onto plates containing test 52°C for 45 s, and 72°C for 45 s, with a final elongation step organisms. The plates were further incubated at 30°C for at 72°C for 10 min. The amplified products were visualized 24 h for bacteria and 48 h for fungi and yeast, and the on a 0.8% (w/v) agarose gel stained with ethidium bromide. zone of inhibition around the isolates was measured PCR products were purified with a PCR product purification (including the diameter of the cylinder, 10 mm). A control kit (Qiagen, Hilden, Germany). The primers used for cylinder containing 15% (w/v) NaCl was used for each sequencing are listed in Coenye et al. (1999). The 16S target microorganism. rRNA sequence has been deposited in the GenBank data library and assigned the accession number HQ918193. The Detection of PKS-I, PKS-II, PKSE, and NRPS sequences sequences obtained were compared with sequences present in the public sequence databases and with EzTaxon, a web- A series of primers was used to detect the genes encoding based tool for the identification of prokaryotes based on 16S the PKS-I and PKS-II, enediyne polyketide synthetase rRNA gene sequences from type strains (Chun et al. 2007). (PKSE), and NRPS genes: The 16S rRNA sequence of strain H16 was aligned using the PKS-I: K1F/M6R (5′-TSAAGTCSAACATCGGBCA- CLUSTAL W (Larkin et al. 2007) against corresponding 3′/5′-CGCAGGTTSCSGTACCAGTA-3′; Ayuso- nucleotide sequences retrieved from GenBank and analyzed Sacido and Genilloud 2005); using the neighbor-joining, maximum-parsimony, and PKS-II: KSα/KSβ (5′-TSGCSTGCTTGGAYGCSATC- maximum-likelihood tools from the PHYLIP package ver. 3′/5′-TGGAANCCGCCGAABCCTCT-3′;Metsä-Ketelä 3.6 (Felsenstein 2005). Phylogenetic trees were visualized et al. 1999); using Dendroscope (Huson et al. 2007). PKSE: EdyA/EdyE (5′-CCGCVCACATCACSGSCC TCGCSGTGAACATGCT-3′/5′- GCAGGCKCCGT DNA–DNA hybridization CSACSGTGTABCCGCCGCC-3′; Liu et al. 2003); NRPS: A3F/A7R (5′-GCSTACSYSATSTACA Cells were disrupted using a French pressure cell (Thermo CSTCSGG-3′/5′-SASGTCVCCSGTSCGGTAS-3′; Spectronic, Rochester, NY), and the DNA in the crude Ayuso-Sacido and Genilloud 2005). lysate was purified by chromatography on hydroxyapatite as described by Cashion et al. (1977). DNA–DNA The 50-μL PCR reaction mixture contained 20–40 ng of hybridization was carried out as described by De Ley et al. DNA template, 0.4 μM of dNTP mixture, 2 μM of each (1970), giving consideration to the modifications described primer, and 10× reaction buffer containing MgCl2, 2.5 U of by Huss et al. (1983), using a model Cary 100 Bio UV/VIS- Taq DNA polymerase and 5% dimethyl sulfoxide. The PCR spectrophotometer equipped with a Peltier-thermostated thermal cycling program included an initial denaturation at 6×6 multicell changer and a temperature controller with 98°C for 4 min, followed by 30 cycles comprising a an in situ temperature probe (Varian Medical Systems, denaturation step at 94°C for 1 min, an annealing step of Palo Alto, CA). 1 min at 57.5°C with K1F/M6R, at 58°C with KSα/ KSβ, at 62°C with EdyA/EdyE, and at 57°C with A3F/ Antimicrobial activity A7R,andterminatedwithanextensionstepof1minat 72°C. A final extension was performed at 72°C for The antagonistic properties of strain H16 were determined 10 min. All of the amplification products were separated by the cylinder plate method against one Gram-positive by electrophoresis in an 0.8% agarose gel, and bands of bacterium (Staphylococcus aureus CIP 53.156), four 1,200–1,400, 600, 1,400, and 700–800 bp were classified Gram-negative bacteria (Acinetobacter baumanii E16, as products of the PKS-I, PKS-II, PKSE, and NRPS genes Enterobacter cloacae E13, Escherichia coli CIP 54.8 T, respectively. 1052 Ann Microbiol (2012) 62:1049–1057

Kinetics of growth and production of antibiotics The UV absorption spectra of the active molecules in methanol were determined with a Shimadzu UV 1605 Fermentations were carried out in ISP-2 (malt extract, 10 g; spectrophotometer (Shimadzu, Kyoto, Japan). The mass yeast extract, 4 g; glucose, 4 g; pH 7.2; distilled water, 1 L) spectra were recorded on an ion-trap mass spectrometer and CMB containing 15% (w/v) NaCl. The pH was (Finnigan, San Jose, CA), equipped with a nanospray adjusted to 7.2 before autoclaving. A seed culture of strain ion electron-spray ionization (nanoESI) source (positive H16 was prepared with the same media and used to ion mode). inoculate 500-mL Erlenmeyer flasks containing 100 mL of medium. The cultures were incubated on a rotary shaker (250 rpm) at 30°C for 14 days. The antimicrobial activities Results and discussion were assayed daily against Klebsiella pneumoniae and Enterobacter cloacae by the agar diffusion method (well Taxonomic characterization of actinomycete strain technique). Each 10-mm-diameter well was filled with 0.2 mL of supernatant. The growth (dry weight of Strain H16 grew well on CMA, nutrient agar (NA), and mycelium) and the pH were also measured. ISP-2 medium and showed little growth on ISP-4 medium. Aerial mycelia were abundant on these first three media, Bioautography and chemical revelation of antibiotics having a white coloration on CMA and NA media and a white-yellow coloration on ISP-2 and ISP-4 media. Substrate Strain H16 was cultured in a 500-mL flask containing mycelia were light brown–olive on CMA and NA media, 100 mL of liquid ISP-2 medium (with 15% w/v NaCl) and bright-yellow and deep brown–yellow on ISP-2 medium, and incubated at 30°C for 5 days. The culture broths were pale yellow on ISP-4 medium. A brownish–yellow diffusible centrifuged to remove cells. The supernatant was extracted pigment was produced on ISP-2 medium. by n-butanol, and the organic extract was concentrated to The substrate mycelia were well developed and frag- dryness. The dry extract was re-dissolved in methanol and mented into rod-like elements. The aerial mycelia were tested by disk diffusion assays. The active extract was irregularly branched and formed straight to flexuous chains applied to a thin-layer chromatography (TLC) silica gel of 10–30 spores per chain. The spores were non-motile, plate (Merck 60 F 254; Merck, Darmstadt, Germany) and smooth-surfaced, and rod-shaped (Fig. 1). developed with an ethanol–ammonia–water mixture The detailed results of the physiological tests are given (8:1:1, v/v/v), as mobile phase. The antibiotics were in Table 1. The strain H16 grew in a wide range of NaCl detected by bioautography (Betina 1973) on silica gel plates seeded with Klebsiella pneumoniae. Clear halos due to growth inhibition of the microorganisms indicated the location of antimicrobial compounds on the TLC plates, and the retention factor (Rf) values were recorded. The antimicrobial compounds were also detected under UV radiation at 254 and 365 nm and by spraying with various chemical reagents, such as naphthoresorcinol–H2SO4, ninhydrin, formaldehyde–H2SO4,andFeCl3, for the detection of sugars, amine groups, polycyclic aromatic, and phenol and hydroxamic acids, respectively (Merck 1975).

Purification and partial characterization of antibiotics

The purification of the antibiotics was performed in a Waters reverse phase high-performance liquid chromatography (HPLC) system using an XBridge C18 (5 μm) column (200 × 10 mm; Waters, Milford, MA) with a continuous linear solvent system consisting of 20–100% methanol in water. The flow rate was 2 mL min-1 and UV detection was at 220 and Fig. 1 Scanning electron micrograph of spore chains of strain H16 275 nm. The final purification was achieved after the second grown on ISP-2 medium supplemented with 15% (w/v) NaCl for re-injection in the HPLC system. 15 days at 30°C. Bar:5μm Ann Microbiol (2012) 62:1049–1057 1053

Table 1 Physiological characteristics of strain H16 The results of the 16S rRNA gene sequence analysis Test Results clearly demonstrated that strain H16 is a member of the genus Actinopolyspora (Fig. 2). The similarity level was NaCl concentration for growth (%, w/v): 98.7% to Actinopolyspora mortivallis DSM 44261T Range 7–32 (Yoshida et al. 1991), the most closely related species. Optimum 15–25 The DNA of strain H16 was hybridized with that of A. pH growth: mortivallis, the closest phylogenetic neighbor, and the Range 5–9 level of DNA–DNA relatedness with this strain was found to Optimum 7 be 87.1% (based on the mean of duplicate determinations, Temperature growth (°C): 83.9 and 90.4%, respectively), which is clearly above the 70% Range 18–50 relatedness guideline proposed by Wayne et al. (1987)for Optimum 30 delineation of separate species. Hydrolysis of: Based on the phenotypic and genotypic results, we concluded Adenine, casein, gelatin, guanine, hypoxanthine, + that strain H16 belonged to the species Actinopolyspora testosterone, Tween 80, and xanthine mortivallis. However, this strain could be distinguished from Starch and tyrosine – A. mortivallis DSM 44261T by a number of phenotypic Nitrate reductase + properties, such as the fragmentation of the substrate mycelium, Utilization of: the degradation of arabinose and raffinose, resistance to Adonitol, arabinose, fructose, galactose, cellobiose, + erythromycin (10 μgmL–1) and penicillin (25 μgmL–1), and erythritol, glucose, glycerol, lactose, maltose, mannose, sensitivity to chloramphenicol (25 μgmL–1). raffinose, ribose, sucrose, trehalose, and xylose sorbitol Inositol, mannitol, melezitose, melibiose, rhamnose, and – salicin Antimicrobial activity Decarboxylation of sodium salts: Acetate, benzoate, butyrate, citrate, oxalate, propionate, – Strain H16 exhibited very strong activity against Klebsiella pyruvate, succinate and tartrate pneumoniae (diameter of inhibition 42 mm) and strong Production of melanoid pigments: activity against Acinetobacter baumanii, Enterobacter On ISP6 and ISP7 media – cloacae,andStaphylococcus aureus (diameters of inhibition Resistance to: 29, 32, and 25 mm, respectively). No activity was observed Chloramphenicol (25 μgmL–1) – against Escherichia coli, Aspergillus niger, Penicillium Erythromycin (10 μgmL–1)+ expansum,orCandida albicans. Earlier studies carried out μ –1 Kanamycin (5 gmL )+by Kokare et al. (2004), Manam et al. (2005), Sarkar et al. μ –1 Penicillin (25 gmL )+(2008), and Huang et al. (2009)alsorevealedthathalophilic μ –1 Streptomycin (10 gmL )+actinomycetes from saline habitats were rich in bioactive +, Property present;-, property absent compounds. Antibiotic production in the genus Actino- polyspora was reported by Kokare et al. (2004)andby Huang et al. (2009). The latter authors described the concentrations (7–32% w/v) and was strictly halophilic. It production of erythronolides in Actinopolyspora sp. used the majority of sugars and other organic compounds, YIM90600, which was later named A. erythraea (Tang et except organic acids, for its growth. It was resistant to most al. 2011). To the best of our knowledge, no antibiotic activity of the antibiotics tested. has been reported in A. mortivallis. Strain H16 contained meso-diaminopimelic acid but not glycine. Whole-cell hydrolysates contained arabinose and Detection of PKS-I, PKS-II, PKSE, and NRPS sequences galactose in addition to ribose, which is typical of cell-wall type IVA (Lechevalier and Lechevalier 1970). The diagnostic Many antibiotics produced by actinomycetes have been phospholipid detected was phosphatidylcholine, corresponding synthesized through a pathway mediated by PKS or/and to phospholipid type PIII (Lechevalier et al. 1977). The NRPS. Strain H16 was analyzed by PCR for the presence predominant fatty acids were saturated branched-chain acids: of PKS-I, PKS-II, PKSE, and NRPS sequences. The latter anteiso- C17:0, (40.3%), iso-C15:0 (14.7%), 9-methyl-C16:0 three genes were detected in this strain (Fig. 3), but the first (13.3%), iso-C17:0 (13.0%), iso-C16:0 (8.9%), and anteiso- gene was absent. PKS-I, PKS-II, PKSE, and NRPS have C15:0 (3.3%). This fatty acid pattern belongs to fatty acid type been detected in several genera of actinomycetes (Ayuso- 2e (Kroppenstedt and Evtushenko 2006). Sacido and Genilloud 2005; Gontang et al. 2010; Janso and 1054 Ann Microbiol (2012) 62:1049–1057

100 H16(HQ918193) 82 Actinopolyspora mortivallis DSM 44261T (DQ883812) Actinopolyspora halophila ATCC 27976T (X54287) Actinopolyspora alba YIM 90480T (GQ480940) 100 Actinopolyspora erythraea YIM 90600T (CQ480939) Saccharomonospora halophila DSM 44411T (AJ278497)

0.01

Fig. 2 Phylogenetic tree derived from 16S rRNA gene sequences showing the position of strain H16 and its phylogenetic neighbors. Bootstrap values >50 % are indicated at nodes. Bar: 0.01 substitutions per nucleotide position. Saccharomonospora halophila is used as the outlier group

Carter 2010), but no studies of these genes in the genus acid (data not published). During the incubation, the pH Actinopolyspora have been reported in the literature. The increased in CM (7.2–8.7) and showed little variation in the genes detected in strain H16 could be involved in the ISP-2 medium (6.5–7.3). activity of this strain against the target microorganisms. Bioautography and partial characterization of antibiotics Kinetics of growth and production of antibiotics The butanolic extract was separated in a TLC system with In liquid media, antibacterial activities against Klebsiella an ethanol–ammonia–water mobile phase. Five active pneumoniae E40 and Enterobacter cloacae E13 were obtained in both ISP-2 and CM (Fig. 4). The antibacterial a P2 CM activity started at the beginning of the exponential phase of 30 growth (2–3 days for K. pneumoniae and 4–5 days for E. 25 cloacae) and reached a maximum after 5 days during the ) –1 mid-stationary phase. This activity was better against K. 20 pneumoniae than against E. cloacae, andE. cloacae E13 15 was found to be more resistant than K. pneumoniae E40 to 10 certain bioactive compounds, such as aztreonam, cefoxitin, (g L Dry cell 5 amoxicillin + clavulanic acid and cefotaxim + clavulanic 0 012345678910 Time (days)

9 8

pH pH 7 6

b P2 CM P2 CM 40 35 30 25 20 15 10 5 Antibacterial activity (mm) activity Antibacterial 0 012345678910 Time (days)

Fig. 4 Time course of growth and pH (a) and antibiotic production Fig. 3 Polymerase chain reaction analysis of strain H16 for nonribosomal (b, based on the inhibition zone against Klebsiella pneumoniae (closed peptide synthases (NRPS) and polyketide synthases (PKS). Lanes: a symbols)andEnterobacter cloacae (open symbols)byActinopolyspora NRPS, b PKS-II, c PKSE, M molecular weight marker (1 kb) mortivallis H16 on ISP-2 (P2)andcomplexmedium(CM) Ann Microbiol (2012) 62:1049–1057 1055

Fig. 5 Nanospray ion electron- spray ionization–mass spectrum of antibiotic E produced by A. mortivallis H16 in positive mode

compounds were detected by bioautography with Rf values enediyne core structure related to glycosylated aromatics. of 0.96, 0.92, 0.75, 0.68, and 0.62; these were designated Our results may suggest the involvement of PKS-II and A, B, C, D, and E, respectively. Compounds B, C, and E PKSE in the synthesis of antibiotics secreted by the strain were strongly active against K. pneumoniae E40 and H16 that contains these genes. However, PKS-II sequences moderately active against E. cloacae E13. Compounds A can also be frequently associated with the production of and D were weakly active and then only against K. pigments in various Streptomyces species (Metsä-Ketelä pneumoniae E40. The five active products showed positive et al. 1999). Our strain also contains the NRPS gene, which chromogenic reactions with formaldehyde–H2SO4 (brown), seems not to have been expressed in the culture medium naphthoresorcinol–H2SO4 (blue), ninhydrin (purple), and used, since we noted the absence of peptidic antibiotics. It is FeCl3 (pink), indicating the presence of polycyclic aromatics, estimated that only 3% of the natural-product potential of sugars, amine groups, and hydroxamic acids respectively. even the well-studied genus Streptomyces has been realized This results suggests that these antibiotics are glycosylated (Watve et al. 2001). polycyclic aromatic compounds containing amine groups In conclusion, our results show that the halophilic strain and hydroxamic acids (Merck 1975). Compounds B, C, and H16 belongs to the species Actinopolyspora mortivallis, but E were purified by HPLC using a semi-preparative C18 differs from the previously described strains by a number of column. The UV-visible spectra of these antibiotics were physiological characteristics and the production of anti- close to each other and showed absorption maxima at 215 biotics. In our study, strain H16 produced glycosylated and 275 nm for B, 218 and 275 nm for C, and 224 and 277 polycyclic aromatic compounds containing amine groups for E. The UV-visible absorption between 275 and 277 nm and hydroxamic acids, both active against Klebsiella suggested the presence of aromatic or heterocyclic com- pneumoniae. These antibiotics could be synthesized pounds. These results are consistent with those obtained in through the biosynthetic pathways involving PKS-II and the chromogenic reactions. The mass spectra obtained by PKSE genes. However, further studies are needed to check nanoESI-mass spectrometry indicated an m/z of 674 for B, this hypothesis. In addition to studying the antibacterial 714 for C, and 884 for E. Figure 5 shows the mass spectrum activity of our newly isolated strain in detail, it would be of compound E in the positive mode. It is known that PKS-II interesting to investigate the anticancer activity of these are involved in the synthesis of aromatic antibiotics secreted antibiotics. Research on the structure and the (Robinson 1991), while PKSE have been implicated in the biosynthesis of these molecules is in progress. synthesis of enediynes. The enediynes, unified by their Acknowledgments We gratefully acknowledge the help of unique molecular architecture and mode of action, represent Gabriele Pötter (DSMZ) for growing and preserving Actinopolyspora some of the most potent anticancer drugs ever discovered mortivallis H16 cultures and for performing chemotaxonomical (Zhang et al. 2008). Several bioactive molecules contain the analyses. 1056 Ann Microbiol (2012) 62:1049–1057

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