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Antagonistic Properties of Some Halophilic Thermoactinomycetes Isolated from Superficial Sediment of a Solar Saltern and Production of Cyclic Antimicrobial Peptides by the Novel Isolate Paludifilum halophilum.

1. Introduction

Actinomycetes are considered as an intermediate group of bacteria and fungi and recognized as prokaryotic organisms. Traditionally, these bacteria have been isolated from terrestrial sources although the first report of mycelium-forming actinomycetes recovered from marine sediments appeared several decades ago [1, 2]. It is only recently that marine-derived actinomycetes have become recognized as a source of novel antibiotics and anticancer agents with unusual structures and properties [3, 4]. However, considering the rising need of new antibiotics to combat the emergence of drug-resistant bacteria, many microbiologists have focused their recent research on actinomycetes from nonconventional environments where particular chemical and physical factors contribute to the selection of species that are best adapted to that extreme environment. To cope with their environmental stressful factors, these microorganisms have developed a complex stress management for their survival, which is being unrevealed for multiple purposes [5, 6]. Accordingly, groups of acidophilic and alkaliphilic, psychrophilic and thermophilic, halophilic and haloalkaliphilic, and xerophilic actinomycetes have been described [7, 8].

In recent years, novel halophilic and halotolerant actinomycetes of diverse genera from diverse families have been isolated from hypersaline environments [9-11]. On the basis of phenotypic, chemotaxonomic, and phylogenetic analysis, several of these halophilic strains were affiliated to the Thermoactinomycetaceae family of the phylum Firmicutes, which was created for the first time in 2006 by Matsuo et al. [12] and included six genera named Thermoactinomyces, Laceyella, Thermoflavimicrobium, Seinonella [13], Planifilum [14], and Mechercharimyces [12]. Recently, numerous novel genera, such as Melghirimyces, Salinithrix, and Croceifilum, were added to this family and the number was extended to seventeen [15, 16]. Except some genera having mesophilic growth below 45[degrees]C, growth in a thermophilic range is a main feature of the Thermoactinomycetaceae family [14]. In addition, several species of the family, such as Melghirimyces algeriensis isolated from an Algerian salt lake [17], Salinithrix halophila from the soil of hypersaline wetland in the north of Iran [18], and Paludifilum halophilum from a superficial sediment of Tunisian solar saltern [16], are halotolerant or halophilic able to support until 20% (w/v) of salinity. Despite the increasing number of halophilic thermoactinomycetes, these microorganisms are still of the least explored ones for novel secondary metabolites. In the field of antimicrobial substances, only some new antibiotics, such as chinikomycin and lajollamycin, are detected in halophilic or halotolerant actinomycete species [4] and several biotechnology companies and academic institutions are currently working on new strategies for the pharmaceutical applications of these new compounds.

Sfax solar saltern, located in the central east of Tunisia, is one of the largest marine salterns in the region. Even though a number of culture-dependent and culture-independent studies were carried out on the biodiversity of eukaryotic [19] and prokaryotic [20, 21] microbial assemblages inhabiting different ponds, there are no reports on any exclusive diversity or biotechnological potential of actinomycetes inhabiting this ecosystem. In a continuous effort to explore the prokaryotic diversity and discover new antimicrobial compounds, we performed a screening procedure to isolate rare halophilic actinomycetes from a concentrator and crystallizer solar saltern ponds and explore their potential to produce drugs against agricultural and human pathogens. The novel isolate Paludifilum halophilum strain SMBg3 with significant antimicrobial activity was characterized further and shown to be potential producer of Gramicidin S and four cyclic antimicrobial dipeptides.

2. Materials and Methods

2.1. Study Site and Samples Collection. The study was conducted in the solar saltern of Sfax located in the central eastern coast of Tunisia (34[degrees]397N and 10[degrees]43,E). It is an artificial ecosystem consisting of a series of interconnected ponds extending over an area of 1500 ha along 12 km of coastline (Figure 1). These ponds are shallow (20-70 cm deep), with a salinity of between 4 and 43% (w/v). The process begins by storing seawater in 17 primary ponds to increase water salinity by evaporation. When the salt concentration reaches the 40-75 g/L range, the seawater is moved to an internal section of five parallel water ponds where it is kept until the salt concentration reaches 130 g/L. After this stage, the seawater is distributed into the six precrystallization ponds to attain a salt concentration of 300 g/L. At the final stage (crystallizer ponds), where the salt precipitates, the brines reach a very high salt concentration (400 to 430 g/L).

Superficial sediment and water samples were taken in December 2012 and February and Mars 2013, from two different salinity ponds, the concentrator pond M1 (salinity 20% (w/v)) and the crystallizer pond TS18 (salinity 38% (w/v)), and immediately stored cold until processing in laboratory within 2 hours of collection. Salinity of the water samples above the sediment was determined at the site with a hand refractometer (ZUZI 5032020), while pH and temperature were measured in situ using, respectively, a digital pH-meter (ISTEK NeoMet pH-220L) and a mercury glass thermometer (Nahita 72075150). Samples for dissolved organic carbon (DOC) were filtered through a 0.22 [micro]m pore size membrane and the concentrations were measured as C[O.sub.2] generated by catalytic combustion using a Shimadzu TOC-V carbon analyzer.

2.2. Isolation of Halophilic Actinomycetes. An aliquot of 1ml of the water sample or 1 g of the superficial sediment (the 0-2 cm fraction) treated with double sonication (Ultrasonic Homogenizers Sonopuls HD 2070) was dispersed in 9 mL of filter sterilized (pore size 0.22 [micro]m) saline water with 15% NaCl. Additional series of dilution were also made and 0.1 mL of the proper dilution was spread on the surface of different selective media, namely, Glucose-Tryptone-Yeast (GTY) [22], Starch Casein Agar (ScA) [23], Bennett medium [24], complex medium (CA) [24], ISP2, and Bergey's Streptomyces medium [25]. Each medium was supplemented with 0.2 [micro]m pore size filtered cycloheximide (25 [micro]g/mL) and nalidixic acid (25 [micro]g/mL) and 15% (w/v) NaCl.

The aerobic development and growth characteristics of halophilic actinomycetes were followed daily at 37[degrees]C on plates and colonies were recognized by their characteristic chalky leather appearance and their severe and dry appearance. After four weeks of growth, colonies were counted and twenty-three, with diverse morphologies, pigmentation, and sizes, were randomly selected from the different media, subcultured several times on their isolation media to obtain pure cultures, and stored at -80[degrees]C in 20% (v/v) glycerol. A code of three letters and one number was assigned for each strain: the first letter of S and W refers, respectively, to the sediment or water origin of the strain; the second letter corresponds to the isolation pond: T (for TS18 pond) and M (for M1 pond); the third letter designates the isolation medium: B (for Bennett medium), G (for GTY medium), Bg (Bergey's medium), C (for CM medium), S (for SCA medium), and I (for ISP2 medium). The final number refers to the number of the isolates.

2.3. In Vitro Antimicrobial Activity. In order to study the antagonistic interaction between environmental actinomycetes, the isolates were grown on Bennett agar plates (10% NaCl) for 21 days at 37[degrees]C. Agar cylinders (6 mm in diameter) were then taken with hollow punch and deposited on the surface of the Bennett agar plate which had previously been seeded with one ml of 4-day cultured target-actinomycete strain. Plates were kept at 4[degrees]C for 2 h and then incubated at 37[degrees]C for 7-14 days. The inhibition zones were measured after incubation and expressed in mm.

The antimicrobial activities of the isolates were also tested against Gram - (Escherichia coli BW25113, Agrobacterium tumefaciens, Salmonella enterica ATCC43972, and Pseudomonas aeruginosa ATCC49189) and Gram+ (Micrococcus luteus LB 14110, Staphylococcus aureus ATCC6538, and Listeria ivanovii BUG 496) bacterial pathogens. The search for antibacterial activity was carried out by the method of disc agar [26], where actinomycete isolates were grown on Bennett agar medium for 14 days at 37[degrees]C and agar cylinders (6 mm in diameter) were then taken and deposited on the surface of the Mueller-Hinton agar plates previously seeded with the test microorganism ([10.sup.5]-[10.sup.6] CFU/mL). The inhibition zones were measured after 24 hours of incubation at 37[degrees]C and expressed in mm.

2.4. Phenotypic and Growth Characteristics of Potential Isolates. Morphological, biochemical, culture, and physiological characterization of potential isolates were determined. Formation of aerial, substrate mycelium and spore arrangements on mycelium were observed with a light microscope (Reichert-Jung series 150 model) and monitored under a phase contrast microscope (Nikon ECLIPSE E600, USA) at 100x magnification. Various colony characteristics such as mycelia color, size, shape, and diffusible pigment production were recorded. Biochemical characterization, namely, Gram reaction, oxidase, catalase, and [H.sub.2]S, and indole production; urease, nitrate, and nitrite reduction; Red Methyl-Voges Prauskuer reactions; ONPG, citrate, and mannitol utilization were also performed as suggested by Holt et al. [27]. NaCl range tolerance and optimal requirement for growth were determined using Bennett medium agar supplemented with different concentrations of NaCl (0,5,10,15,20,25, and 30%). Temperature and pH range for growth were also determined using Bennett medium [28].

2.5. DNA Extraction of Potential Isolates and PCR Amplification of 16S rRNA. The six potential isolates were grown for 4 days at 37[degrees]C with agitation in 15 ml of Bennett medium. Biomass was harvested by centrifugation at 4,000 rpm for 15 min and washed twice with sterile saline water. The method of Rainey et al. [29] was used for the extraction and purification of genomic DNA. The 16S rDNA gene of the six isolates was amplified by polymerase chain reaction (PCR) using primers fD1 (5'AGAGTT TGATCCTG GCTCAG 3') and Rs16 (5'AAG GAG GTG ATC CAA GCC 3') [30]. The final volume of the reaction mixture of 50 [micro]l contained tampon buffer (10x) with 50 mM Mg[Cl.sub.2], deoxynucleoside triphosphates (10 mM dNTP), 10 [micro]M (each) S1 and S2 primers, 2 [micro]L (80 ng) DNA, and 0.1 [micro]L Taq DNA polymerase (5U/[micro]L). Amplification was made using a Basic PCR protocol which consisted of an initial denaturation at 95[degrees]C for 10 min, followed by 30 amplification cycles of 94[degrees]C for 45 s, 52[degrees]C for 30 s, and 72[degrees]C for 1min and 30 s and a final extension step of 72[degrees]C for 10 min [31]. The amplification result was detected by agarose gel (1%) electrophoresis and visualized by ultraviolet fluorescence after ethidium bromide staining [32]. The purification of DNA 16S fragment from PCR on agarose gels was performed using the PureLink Quick Gel Extraction and PCR Purification Combo Kit. The same primers were then used separately in two sequencing reactions from the two ends of the amplified fragment (about 1.5 kbp). The two sequences obtained were compared for similarity with those contained in genomic database banks, using the NCBI BLAST [33].

2.6. Phylogenetic Analysis. Sequence data were established with the BioEdit program (http://www.mbio.ncsu.edu/ BioEdit/bioedit.html) and studied for sequence homology with the archived 16S rDNA sequences from GenBank at https://www.ncbi.nlm.nih.gov/nucleotide, using the BLAST search program [34]. Different sequences were aligned with CLUSTAL W [35,36] and a phylogenetic tree was constructed using the neighbor-joining DNA distance algorithm within the MEGA6 (Molecular Evolutionary Genetics Analysis Version 6.0) (http://www.megasoftware.net/) software [37]. The 16S rRNA gene sequences of the six potential thermoactinomycete strains have been deposited in the GenBank database under the accession numbers of KP229518KP229523.

2.7. Production, Extraction, and Liquid Chromatography-High Resolution Mass Spectrometry Analysis of Antimicrobial Products from Paludiflum halophilum. Paludiflum halophilum strain SMBg3 was cultured on Bennett medium supplemented with 10% NaCl, for 7 days at 37[degrees]C. Mycelium was scraped and inoculated into four Erlenmeyer flasks (1L) containing 250 mL of the same medium. After seven days of incubation, the total broth was mixed, centrifuged at 4000g for 15 min, and then filtered through Whatman number 1 filter paper. The pellet was transferred aseptically into a conical flask and an equal volume of ethyl acetate was added to the filtrate and shaken vigorously for 2 hours for the complete extraction of the antibacterial compounds. The ethyl acetate phase containing the active principal was separated from the aqueous phase and was evaporated to a residue using Rota vapor (Heidolph: P/N Hei-VAP Value/G3: 560-01300-00). One mg of the residue was accurately weighed and dissolved in 300 [micro]L of ethyl acetate and this solution was filtered through 0.2 [micro]m PTfE filter into HPLC vial where it is submitted to LC/MS analysis. High resolution mass spectrometric data were obtained using a ThermoLTQ-Orbitrap coupled to an HPLC system (PDA detector, PDA autosampler, and pump). The following conditions were used: capillary voltage of 45 V, capillary temperature of 260[degrees]C, auxiliary gas flow rate of 10-20 arbitrary units, sheath gas flow rate of 40-50 arbitrary units, spray voltage of 4.5 kV, and mass range of 100-2000 amu (maximal resolution of 30000). For LC/MS, a Sunfire C18 analytical HPLC column (5 [micro]m, 4.6 mm x 150 mm) was used with a mobile phase of 0 to 100% MeOH over 30 min at a flow rate of 1 mL/min.

3. Results and Discussion

3.1. Physicochemical and Microbiological Analysis of Samples. The physicochemical parameters of the water above the sediment surface and the microbiological parameters of the water and superficial sediment from which cores were collected during the three campaigns are summarized in Table 1. M1 pond was characterized by an intermediate salinity ranging from 19 to 21%, whereas the TS18 pond had a higher salinity that varied between 31 and 36%. Temperature was slightly higher in pond TS18 than in M1, while pH was slightly alkaline in M1 and close to neutrality in TS. Moreover, dissolved organic carbon was found to be significantly higher in TS18 than in M1. It was noticed that superficial sediment samples from M1 contained gypsum deposit, while those of TS were constituted with halite. The maximum values of total cell counts (5.9 x [10.sup.9] cells/g) were detected in the sediment of the TS18 pond and were 2-3-fold higher than in M1 pond (Table 1). In addition, when comparing water and sediment samples, it was found that, for both prospected ponds, the maximum values of total cell counts were detected in sediments and were 3- to 10-fold higher than those obtained in waters (Table 1). This could be attributed to greater organic matter abundance [38] and lower predation rates [19, 39].

The cultivable actinomycete density during the 3 sampling campaigns showed different patterns in M1 and TS18 ponds, with counts between 2.5 and 7 times higher in TS18 than in M1 (Table 1). Intriguingly, for both M1 and TS18, no cultivable actinomycetes could be detected in waters, while their counts in superficial sediments were much lower than those reported in previous studies [40, 41].This could be explained, in part, by the high salinity of the prospected ponds and the severe selective pressure of our isolation procedure, which allows the growth of only halophilic strains able to support at least a salinity of 15%. In fact, the occurrence of actinomycetes in the hypersaline environment has been reported in several previous studies, stating the decrease in actinomycetes colonies forming units counts with the increase in salinity [41, 42]. In addition, the ability of actinomycete cells to enter a viable but nonculturable state in response to stressful conditions, in which bacteria lost their ability to form colonies in the surface of solid media, could not be discarded [43, 44].

3.2. Isolation of Strains and Screening of Antimicrobial Activities. Given that less than 1% of bacteria from saline environments can be cultured, the use of appropriate isolation media is critical for improving the recovery of Actinobacteria [45]. Six isolation media were chosen in this study to select for Actinobacteria. During the 3 sampling campaigns and based on colony morphology, growth characteristics, and macroscopic examination, a total of twenty-three actinomycete strains were collected on all isolation media with 3 being isolated from Ml and 20 from TS18 (Table 1). Bennett medium exhibited the highest recovery producing 9 isolates, followed by GTY medium with 7 strains while no strain was recovered on ISP2 medium. Most isolates showed, on Bennett medium at 10% NaCl, aerial mycelia with color varied from yellow and light yellow to beige white and fluorescent spores arranged in chains.

The 23 strains were tested for their ability to produce antimicrobial substances. The result of antagonistic interactions between the actinomycete strains, taken in pairs, allowed the detection of 18 active strains named STS3, STB1, STG6, STG1, STC3, STS2, STC4, STG4, STB7, SMC3, SMBg3, STG8, STC5, STG2, STB6, STB8, STB2, and STG5 producing antimicrobial compounds against one or more targetactinomycete strains (Table 2). The halo diameter was used to monitor each strain level of antimicrobial substance produced. Our results showed that the inhibition zones diameter versus the target strain ranged from 13 to 33 mm and their activity spectrum comprised between one and 8 target strains, which suggested that the produced substances could be of a different nature (Table 2). Most strains (thirteen of the eighteen) have a wide spectrum of inhibition, with at least two sensitive strains (Table 2).

The isolates antibacterial potential was also analyzed against seven pathogen strains and the antibacterial activity extent was varied among the actinobacterial isolates (Table 3). Nineteen out of the 23 strains of halophilic actinomycetes exhibited appreciable inhibitory activity against Gram-negative and/or Gram-positive bacteria. Among them, 5 strains, named STB2, STC3, STG2, SMB5, and SMC3, acted only against Gram-negative bacteria and 6 strains (STG3, STG4, STG6, STB1, STB3, and STB4) against only Grampositive bacteria. The 8 remaining strains (STB6, STB8, STS2, STS3, STC5, STC4, STG1, and SMBg3) revealed excellent antibacterial activity against both Gram-positive and Gram-negative bacteria. Interestingly, when the antagonistic activity was lacking for one strain, either it was completely inactive on human pathogens or its activity was strongly reduced. In the literature, while the antagonistic properties of halotolerant and moderate halophilic actinomycetes have often been reported in the literature at low or medium salinity [41, 46, 47], those of halophilic isolates at salinity close to saturation have never been mentioned. This is the first report showing that the crystallizer and noncrystallizer ponds of Sfax marine saltern harbored potential halophilic actinomycetes producing antimicrobial compounds against Gram-positive and Gram-negative pathogen bacteria. Based on their broad (STB8, STS2, STS3, STC4, and SMBg3) or narrow (SMC3) activity spectrum, 6 strains among all the actinomycete isolated were, therefore, subjected to detailed taxonomic studies.

3.3. Characterization of Potential Isolates. In order to estimate the relatedness between the 6 potent isolates, the physiological, biochemical, and growth characteristics of each strain were compared. Results in Table 4 revealed that the colonies of the six isolates were circular and aerial mycelium was observed for only STC4, SMC3, STB8, and SMBg3 with fluorescent spores arranged in chains. However, substrate mycelium was between pale yellow for SMBg3 and SMC3, white for STS3 and STS2, colorless for STC4, and transparent for STB8. Gram and catalase reactions were positive for all strains. All the isolates could not metabolize mannitol and ONPG or produce [H.sub.2]S. Only STC4 and STB8 strains were nitrate reductase +, while RM and VP reactions were negative for all isolates, except STB8 which was RM positive. Isolates were also screened for their growth at various NaCl, temperature, and pH levels. All isolates exhibited growth in the NaCl range of 5-20% with an optimum at 10% NaCl and at a temperature range of 30-55[degrees]C with an optimum of 45[degrees]C, while pH range for growth was between 5.0 and 11.0 with an optimum at 8.0-8.5 for all strains. These results showed that all isolates in this study were halophilic and thermotolerant. Carbon source utilization is also given in Table 4, showing that the six strains could metabolize glucose and starch, but not sucrose. However, saccharose, maltose, and xylose were metabolized weakly by STS2 strain and strongly by the others. Our results showed also that the actinomycete strains constitute potential producers of amylase (69% of the total isolates), followed by protease (52%), cellulase, DNase (39%), and lipase (4%). This is in agreement with our previous studies conducted on the same ponds which found that the most frequent hydrolytic activity among archaeal isolates was observed for amylase and protease [21]. Assuming that these frequencies are related to the nature of the organic matter in the pond, these results may suggest that carbohydrates and proteins in the sediment are the major carbon sources for the halophilic prokaryotes inhabiting the two ponds [19].

To ascertain the phylogenetic relationships of potential strains, their almost-complete 16S rRNA gene sequences (1352-1483 bp) were determined. A comparative sequence analysis using the BLAST program and a phylogenetic analysis using neighbor-joining revealed that the six strains were very close and formed a distinct sublime within the Thermoactinomycetaceae family (Figure 2). The six strains shared the highest 16S rRNA sequence similarity with respect to the strain types of Salinithrix halophila CECT [8506.sup.T] (94%), Desmospora activa DSM 45169t (94%), Kroppenstedtia guangzhouensis KCTC [29149.sup.T] (95%), Kroppenstedtia eburnea DSM [45196.sup.T] (95%), and Melghirimyces algeriensis (95%). These similarity percentages lower than 97% suggested that the isolated actinomycete strains could represent new members of the Thermoactinomycetaceae family and their sequences were published in the GenBank database under the accession numbers of KP229518-KP229523. To go further in the taxonomic position of these strains in the phylum Actinobacteria, we recently performed a polyphasic taxonomic study on strain SMBg3 [16]. Our results revealed that this strain occupied an independent phylogenetic lineage distinct from all other reference genera within the family Thermoactinomycetaceae. On the basis of these data and other phenotypic and chemotaxonomic characteristics, strain SMBg3 was assigned to a new genus in the family Thermoactinomycetaceae for which the name Paludifilum was proposed and the type strain of the type species [SMBg3.sup.T] was named Paludifilum halophilum and deposited in the DSMZ(=[DSM102817.sup.T]) and CCUG(=CCUG [68698.sup.T]) public collections [16]. In fact, the occurrence of new genera of halophilic thermoactinomycetes has been reported in the hypersaline environments of distant geographical sites such as the Algerian salt lake [17] and the soil of hypersaline wetland in the north of Iran [18]. The saltern of Sfax can thus be considered as an additional geographical site harboring new members of halophilic thermoactinomycetes. Moreover, this is the first report revealing that these microbes could have an antimicrobial potential.

3.4. Production, Purification, and LC/MS Characterization of Antimicrobial Compounds Produced by Paludifilum halophilum. In order to unveil the preliminary characteristics of antimicrobial compounds produced by Paludifilum halophilum strain SMBg3, the strain was cultivated in large scale and active substances were extracted by ethyl acetate as mentioned above and tested against three Gram-positive and four Gram-negative bacteria at a 2.5mg/disc concentration. Our results showed good activity against S. aureus ATCC6538 (14 mm), E. coli W 25113 (15 mm), A. tumefaciens (14 mm), S. enterica ATCC43972 (16 mm), and P. aeruginosa ATCC49189 (16 mm). No inhibition was observed for M. luteus and L. ivanovii. In a previous study by Vijayakumar et al. [48], the ethyl acetate extract of Streptomyces sp. was highly active against Vibrio cholerae (26 mm), Salmonella typhi (24 mm), Proteus vulgaris (23 mm), Candida albicans (17 mm), Klebsiella pneumoniae (16 mm), Proteus mirabilis (15 mm), Staphylococcus aureus (15 mm), and Escherichia coli (14 mm). In Tunisia, little attention has been paid to the antimicrobial activity of actinomycetes. To date, only one study has been conducted by Trabelsi et al. [49] on different rhizospheric soils and showed that, among fifty-four isolates of actinomycetes collected, 42 strains were classified as Streptomyces, 4 strains were classified as Micromonospora, 1 was classified strain as Pseudonocardia, 1 strain was classified as Actinomadura, 1 was classified strain as Nocardia, and 5 strains were classified as non-Streptomyces. In addition, more than the half of the ethyl acetate extract of these isolates was shown to inhibit at least one tested pathogenic microorganisms in liquid culture.

The LC-MS profiles of the crude extract of Paludifilum halophilum strain SMBg3 are shown in Figure 3. The chromatograph showed five major constituents whose structures are provided in Table 5. Molecular formula and chemical structures of these compounds were deduced from the quasimolecular ion peak [M + H]+ on the basis of their HRESI-MS spectrum. These five major constituents are the cyclic lipopeptide Gramicidin S (Rt: 9.08 min) and four cyclic dipeptides (CDPs) identified as cyclo(L-4-OH-Pro-L-Leu) (Rt: 4.08 min), cyclo(L-Tyr-L-Pro) (Rt: 4.83 min), cyclo(LPhe-L-Pro) (Rt: 5.18 min), and cyclo(L-Leu-L-Pro) (Rt: 5.87 min). In fact, CDPs [also known as 2,5-dioxopiperazines; 2,5-diketopiperazines; cyclo(dipeptides); or dipeptide anhydrides] are well known for their economically beneficial biological activities and therefore are among the most common peptide derivatives found in nature [50]. They have been isolated from microorganisms, sponges, and from a variety of tissues and body fluids [51-53]. The cyclic form of the dipeptide is often more stable in vivo than its linear counterpart, making them far more promising in terms of drug candidacy [54]. Both natural and synthetic diketopiperazines have a wide variety of biological activities, including antitumor [55] (Nicholson et al. 2006), antiviral [56], antifungal [57], and antibacterial [58] activities. For instance, the cyclo(Pro-Tyr) was first isolated from Alternaria alternate [53] and then from Streptomyces sp. TN25644. Cain et al. [59] reported that cyclo(Pro-Tyr) exhibits no activity against strains of Micrococcus luteus, Mycobacterium smegmatis, Sacharomyces cerevisiae, Candida neoformans, Candida albicans, and Aspergillus niger. In contrast, Smaoui et al. [60] reported a strong antimicrobial activity of cyclo(Pro-Tyr) against Micrococcus luteus LB 14110, Salmonella enterica ATCC43972, and Fusarium sp.

4. Conclusions

This study deals with the isolation, characterization, and antimicrobial potentiality of a collection of halophilic actinomycete strains isolated from the solar saltern of Sfax. Our results revealed, for the first time, that the superficial sediment of this ecosystem is a source of novel halophilic actinomycetes belonging to the Thermoactinomycetaceae family with unexplored potential for antimicrobial discovery. For the potent strain SMBg3, assigned to a new genus in the family and named Paludifilum halophilum, we have shown that the observed antimicrobial activity is most likely explained by the production of Gramicidin S and four cyclic dipeptides identified as cyclo(L-4-OH-Pro-LLeu), cyclo(L-Tyr-L-Pro), cyclo(L-Phe-L-Pro), and cyclo(LLeu-L-Pro). The potent inhibitory effect of these compounds covered the growth of the plant pathogen A. tumefaciens and the human pathogens S. aureus, S. enterica, E. coli, and P. aeruginosa. To the best of our knowledge, this is the first time that the bioactivity of cyclic antimicrobial peptides from halophilic thermoactinomycete against agriculturally and medically important bacteria is reported. Our work is now in progress to purify these cyclic antimicrobial peptides for further characterization.

https://doi.org/10.1155/2017/1205258

Conflicts of Interest

The authors declare no conflicts of interest.

Acknowledgments

The authors are grateful to Dr. Ridha Amdouni from COTUSAL salt company for allowing members of their research team access to the saltern and Dr. Hafeth Bjaoui from the English Language Unit at the Faculty of Science of Sfax, Tunisia, for careful proofreading.

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Donyez Frikha Dammak, (1) Ziad Zarai, (2) Soumaya Najah, (3) Rayed Abdennabi, (4) Lassaad Belbahri, (4) Mostafa E. Rateb, (5) Hafedh Mejdoub, (6) and Sami Maalej (1)

(1) Unite Biodiversity et Ecosystemes Aquatiques Environnementaux (UR/UES/72), Faculte des Sciences de Sfax, Universite de Sfax, BP 1171, 3000 Sfax, Tunisia

(2) Laboratoire de Biochimie et de Genie Enzymatique des Lipases, ENIS, BPW, 1173 Sfax, Tunisia

(3) Institut de Biologie Integrative, UMR 9198, Universite Paris-Sud, Bat 400, 91405 Orsay Cedex, France

(4) Laboratory of Soil Biology, University of Neuchatel, 11 Rue Emile Argand, 2000 Neuchatel, Switzerland

(5) School of Science & Sport, University of the West of Scotland, Paisley PA12BE, UK

(6) Laboratoire des Biotechnologies Vegetales Appliquees a 'melioration des Cultures, FSS, Universite de Sfax, BP 1171, 3000 Sfax, Tunisia

Correspondence should be addressed to Sami Maalej; sami.maalej@fss.rnu.tn

Received 23 March 2017; Accepted 18 June 2017; Published 27 July 2017

Academic Editor: Pedro J. Garcia-Moreno

Caption: Figure 1: Map of the location of the two sampling ponds (TS18 and M1) of the Sfax solar saltern.

Caption: Figure 2: Neighbor-joining tree based on 16S rDNA sequences showing the relations between the halophilic actinomycete strains (STS2, STC4, STB8, SMBg3, STC3, andSTS3) and type species of the family Thermoactinomycetaceae. The accession numbers of strain sequences are given in parentheses. The numbers at the nodes indicate the levels of bootstrap support based on neighbor-joining analyses of 1000 resampled datasets; only values over 50% are given. Bar: 0.02 nucleotide substitutions per nucleotide position. Staphylococcus aureus (ATCC14458) is given as outgroup.

Caption: Figure 3: LC/MS chromatogram of the crude extract from Paludifilum halophilum strain SMBg3.
Table 1: Range of physicochemical characteristics of water above the
two ponds' sediment surface and microbiological parameters during the
three campaigns.

Parameters                                             Pond M1

Physicochemical
pH                                               8.3-8.5 [+ or -]0.1
Salinity (%)                                       19-21 [+ or -] 1
Temperature ([degrees]C)                           14-19 [+ or -] 1
Dissolved organic carbon (mg [l.sup.-1])         2.7-3.1 [+ or -] 0.5
Microbiological
Water total cell count ([10.sup.8] cells        1.2-1.8 [+ or -] 0.01
   [ml.sup.-1])
Sediment total cell count ([10.sup.8] cells       13-19 [+ or -]0.02
   [g.sup.-1])
Water cultivable actinomycete count (UFC                  0
   [ml.sup.-1])
Sediment cultivable actinomycete count             3-30 [+ or -] 1
   (UFC [g.sup.-1])
Number of actinomycete isolates from water                0
Number of actinomycete isolates from                      3
   sediment

Parameters                                            Pond TS18

Physicochemical
pH                                               7.2-7.8 [+ or -] 0.2
Salinity (%)                                       31-36 [+ or -] 1
Temperature ([degrees]C)                           16-22 [+ or -] 1
Dissolved organic carbon (mg [l.sup.-1])         5.8-7.4 [+ or -] 0.5
Microbiological
Water total cell count ([10.sup.8] cells         3.2-19 [+ or -] 0.05
   [ml.sup.-1])
Sediment total cell count ([10.sup.8] cells       36-59 [+ or -] 0.1
   [g.sup.-1])
Water cultivable actinomycete count (UFC                 0-1
   [ml.sup.-1])
Sediment cultivable actinomycete count             20-70 [+ or -] 3
   (UFC [g.sup.-1])
Number of actinomycete isolates from water                0
Number of actinomycete isolates from                      20
   sediment

Each data is mean of three independent analyses [+ or -] standard
deviation; P value < 0.05.

Table 2: Antagonistic interactions between the isolate strains.

Producer                       Target strain
strain
              STS2     STG1     STB6     STG5     STB8     STG4
STG3
STS3           13       18       --       28       --       18
STB1           --       --       --       --       --       --
STBS           --       --       --       --       --       --
STB4           --       --       --       --       --       --
STG6           --       --       --       --       --       --
STG1           --       --       12       --       --       17
STC3           --       29       --       --       --       --
STC4           14       34       14       --       23       --
STG5           --       --       15       --       --       17
STB6           --       --       --       17       --       --
STB8           16       --       13       --       --       14
STB2           13       --       --       --       14       12
STS2           --       22       --       --       --       14
STG4           --       --       --       --       --
STB7           --       --       18       --       --       --
STC5           --       25       --       --       --       --
SMC3           --       --       --       --       --       --
STG2           --       --       --       --       --       --
STG8           --       --       --       --       --       --
STB9           --       --       --       --       --       --
SMBg3          --       --       --       --       --       --
SMB5           --       --       --       --       --       --

Producer                       Target strain
strain
              STC5     STG8     STB7     STG6     STG3     STG2
STG3
STS3           --       --       --       --       --       18
STB1           --       --       --       --       --       15
STBS           --       --       --       --       --       --
STB4           --       --       --       --       --       --
STG6           --       --       --       --       --       --
STG1           --       --       --       --       --       22
STC3           --       --       --       --       --       --
STC4           --       --                --       --       --
STG5           --       --       12       --       --       19
STB6           --       --       --       --       --       --
STB8           19       --       --       18       --       --
STB2           --       --       --       --       --       --
STS2           --       --       --       --       --       --
STG4           --       --       --       --       --       --
STB7           --       --       --       --       --       --
STC5           --       --       --       --       --       --
SMC3           --       --       --       --       --       --
STG2           --       --       --       --       --       --
STG8           --       --       --       --       --       --
STB9           --       --       --       --       --       --
SMBg3          --       --       --       --       --       --
SMB5           --       --       --       --       --       --

Producer                       Target strain
strain
              STS3     STC4     STB2     STC3     STB1     STBS
STG3
STS3           --       --       --       --       --       --
STB1           --       --       --       --       --       --
STBS           --       --       --       --       --       --
STB4           --       --       --       --       --       --
STG6           --       --       --       --       --       --
STG1           15       --       15       --       --       --
STC3           --       --       --       --       --       --
STC4           --       --       --       --       --       --
STG5           17       --       --       --       --       --
STB6           --       --       --       --       --       --
STB8           --       --       --       --       --       --
STB2           --       --       --       --       --       --
STS2           --       --       --       --       --       --
STG4           --       --       --       --       --       --
STB7           2        --       --       --       --       --
STC5           15       --       --       --       --       --
SMC3           --       --       --       --       --       --
STG2           --       --       --       --       --       --
STG8           --       --       --       --       --       --
STB9           --       --       --       --       --       --
SMBg3          --       --       --       --       --       --
SMB5           --       --       --       --       --       --

Producer                    Target strain
strain
              STB4     SMC3     STB9     SMB5    SMBg3
STG3
STS3           --       --       14       16       --
STB1           --       --       --       --       --
STBS           --       --       --       --       --
STB4           --       --       --       --       --
STG6           --       --       23       25       --
STG1           --       14       --       --       --
STC3           --       --       16       22       --
STC4           --       15       16       18       --
STG5           --       13       16       18       --
STB6           --       --                --       --
STB8           --       --       22       17       16
STB2           --       --       17       12       --
STS2           --       --       --       --       --
STG4           --       --       24       27       --
STB7           --       17       --       15       --
STC5           --       --       16       17       --
SMC3           --       --       --       18       --
STG2           --       --       18       --       --
STG8           --       --       16       --       --
STB9           --       --       --       --       --
SMBg3          --       --       32       33       --
SMB5           --       --       --       --       --

Results shown in the above table are average of triplicate parallel
experiments.

Table 3: Antimicrobial activities of halophilic actinomycetes isolated
from solar salterns of Sfax.

Strains     A. tumefaciens    S. aureus    S. enterica    M. luteus

STB2              --              --            --            --
STB6              --              --            14            --
STB8              12              11            16            --
STG5              --              --            --            --
STS2              14              --            12            12
STS3              15              12            14            --
STC4              12              14            14            14
STC5              --              --            --            --
STC3              --              --            --            --
STG3              --              --            --            --
STG4              --              23            --            --
STG6              14              --            --            --
STB1              --              --            --            --
STB3              --              --            --            --
STB4              --              --            --            --
STG8              --              --            --            --
STG1              15              --            --            12
STB7              --              --            --            --
STG2              --              --            16            --
STB9              --              --            --            --
SMBg3             15              15            16            --
SMB5              --              --            --            --
SMC3              --              --            --            --

Strains     E. coli     L. ivanovii    P. aeruginosa

STB2           12            --              15
STB6           17            13              16
STB8           14            --              21
STG5           --            --              --
STS2           16            14              --
STS3           17            --              --
STC4           14            --              --
STC5           15            12              --
STC3           13            --              19
STG3           --            11              --
STG4           --            12              --
STG6           --            13              --
STB1           --            11              --
STB3           --            11              --
STB4           --            11              --
STG8           --            --              --
STG1           15            --              --
STB7           --            --              --
STG2           15            --              --
STB9           --            --              --
SMBg3          15            --              16
SMB5           20            --              --
SMC3           13            --              --

Results shown in the above table are average of triplicate parallel
experiments.

Table 4: Phenotypic characteristics of potential actinomycete
isolates.

Characteristics                            STS2

Pond/growth medium                        TS/SCA
Morphological characteristics
Colony aspect                            Circular
Colony size (mm)                            4-8
Aerial mycelium                           Absent
Substrate mycelium                         White
Spore chains/fluorescence        Short chains/fluorescent
Biochemical characteristics
Gram staining                                +
Catalase                                     +
Oxidase
Mannitol                                     -
Citrate                                      -
H2S production                               -
Nitrate reductase                            -
Nitrite reductase                            -
ONPG                                         -
Urea                                         -
Indole production                            -
RM                                           -
VP                                           -
Growth characteristics
% NaCl range (optimum)                   5-15 (10)
Temperature range (optimum)              30-55(45)
pH range (optimum)                       5-11 (8)
Carbon source utilization (1%)
Glucose                                      +
Sucrose                                      -
D(+) Saccharose                             +/-
D Maltose                                   +/-
D-Xylose                                    +/-
Mannitol                                     -
Starch                                       +

Characteristics                              STS3

Pond/growth medium                          TS/SCA
Morphological characteristics
Colony aspect                              Circular
Colony size (mm)                             4-8
Aerial mycelium                             Absent
Substrate mycelium                          White
Spore chains/fluorescence        Long chains/very fluorescent
Biochemical characteristics
Gram staining                                 +
Catalase                                      +
Oxidase
Mannitol                                      -
Citrate                                       -
H2S production                                -
Nitrate reductase                             -
Nitrite reductase                             -
ONPG                                          -
Urea                                          -
Indole production                             -
RM                                            -
VP                                            -
Growth characteristics
% NaCl range (optimum)                    5-20 (10)
Temperature range (optimum)               30-55(45)
pH range (optimum)                         5-11 (8)
Carbon source utilization (1%)
Glucose                                       +
Sucrose                                       -
D(+) Saccharose                               +
D Maltose                                     +
D-Xylose                                      +
Mannitol                                      +
Starch                                        +

Characteristics                            STC4

Pond/growth medium                        TS/SCA
Morphological characteristics
Colony aspect                            Circular
Colony size (mm)                            2-4
Aerial mycelium                           Yellow
Substrate mycelium                       Colorless
Spore chains/fluorescence        Short chains/fluorescent
Biochemical characteristics
Gram staining                                +
Catalase                                     +
Oxidase
Mannitol                                     -
Citrate                                      -
H2S production                               -
Nitrate reductase                            +
Nitrite reductase                            -
ONPG                                         -
Urea                                         -
Indole production                            -
RM                                           -
VP                                           -
Growth characteristics
% NaCl range (optimum)                   5-20 (10)
Temperature range (optimum)              30-55(45)
pH range (optimum)                       5-11 (8)
Carbon source utilization (1%)
Glucose                                      +
Sucrose                                      -
D(+) Saccharose                              +
D Maltose                                    +
D-Xylose                                     +
Mannitol                                     +
Starch                                       +

Characteristics                            SMBg3

Pond/growth medium                         Ml/Bg
Morphological characteristics
Colony aspect                            Circular
Colony size (mm)                            4-8
Aerial mycelium                         Pale yellow
Substrate mycelium                      Yellow pale
Spore chains/fluorescence         Long chains/fluorescent
Biochemical characteristics
Gram staining                                +
Catalase                                     +
Oxidase
Mannitol                                     -
Citrate                                      -
H2S production                               -
Nitrate reductase                            -
Nitrite reductase                            -
ONPG                                         -
Urea                                         -
Indole production                            -
RM                                           -
VP                                           -
Growth characteristics
% NaCl range (optimum)                   5-20 (10)
Temperature range (optimum)              30-55(45)
pH range (optimum)                       5-11 (8)
Carbon source utilization (1%)
Glucose                                      +
Sucrose                                      -
D(+) Saccharose                              +
D Maltose                                    +
D-Xylose                                     +
Mannitol                                    +/-
Starch                                       +

Characteristics                            SMC3

Pond/growth medium                         Ml/CA
Morphological characteristics
Colony aspect                            Circular
Colony size (mm)                            4-8
Aerial mycelium                           Yellow
Substrate mycelium                      Yellow pale
Spore chains/fluorescence        Short chains/fluorescent
Biochemical characteristics
Gram staining                                +
Catalase                                     +
Oxidase
Mannitol                                     -
Citrate                                      +
H2S production                               -
Nitrate reductase                            -
Nitrite reductase                            -
ONPG                                         -
Urea                                         +
Indole production                            +
RM                                           -
VP                                           -
Growth characteristics
% NaCl range (optimum)                   5-20 (10)
Temperature range (optimum)             30-55 (45)
pH range (optimum)                       5-11 (8)
Carbon source utilization (1%)
Glucose                                      +
Sucrose                                      -
D(+) Saccharose                              +
D Maltose                                    +
D-Xylose                                     +
Mannitol                                     +
Starch                                       +

Characteristics                            STB8

Pond/growth medium                         TS/Bt
Morphological characteristics
Colony aspect                            Circular
Colony size (mm)                            1-5
Aerial mycelium                           Yellow
Substrate mycelium                      Transparent
Spore chains/fluorescence         Long chains/fluorescent
Biochemical characteristics
Gram staining                                +
Catalase                                     +
Oxidase
Mannitol                                     -
Citrate                                      -
H2S production                               -
Nitrate reductase                            +
Nitrite reductase                            -
ONPG                                         -
Urea                                         +
Indole production                            -
RM                                           +
VP                                           -
Growth characteristics
% NaCl range (optimum)                   5-15 (10)
Temperature range (optimum)              30-55(45)
pH range (optimum)                       5-11 (8)
Carbon source utilization (1%)
Glucose                                      +
Sucrose                                      -
D(+) Saccharose                              +
D Maltose                                    +
D-Xylose                                     +
Mannitol                                     +
Starch                                       +

Table 5: LC-HR-ESI-MS analysis of the most interesting compounds
extracted from liquid cultures of Paludifilum halophilum strain
SMBg3.

Rt (min)     Accurate                    Molecular
               mass                      formula *

9.08        1141.71463    [C.sub.60][H.sub.92][O.sub.10][N.sub.12]
4.08         22713956      [C.sub.11][H.sub.18][N.sub.2][O.sub.3]
4.83        261.12405      [C.sub.14][H.sub.16][N.sub.2][O.sub.3]
5.18        245.12918      [C.sub.14][H.sub.16][N.sub.2][O.sub.2]
5.87        211.14438      [C.sub.11][H.sub.18][N.sub.2][O.sub.2]

Rt (min)      Suggested compound          Notes

9.08           Gramicidin S-1141       Lipopeptide
4.08       Cyclo(L-4-OH-Pro- L-Leu)     Dipeptide
4.83          Cyclo(L-Tyr-L-Pro)        Dipeptide
5.18          Cyclo(L-Phe-L-Pro)        Dipeptide
5.87          Cyclo(L-Leu-L-Pro)        Dipeptide

Rt (min)                   Structure

9.08       [mathematical expression not reproducible]
4.08       [mathematical expression not reproducible]
4.83       [mathematical expression not reproducible]
5.18       [mathematical expression not reproducible]
5.87       [mathematical expression not reproducible]

* Formula deduced from the quasimolecular ion peak [[M + H].sup.+].
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Title Annotation:Research Article
Author:Dammak, Donyez Frikha; Zarai, Ziad; Najah, Soumaya; Abdennabi, Rayed; Belbahri, Lassaad; Rateb, Most
Publication:BioMed Research International
Article Type:Report
Date:Jan 1, 2017
Words:8179
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