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New dithiolopyrrolone antibiotics induced by adding sorbic acid to the culture medium of Saccharothrix algeriensis NRRL B-24137

Rabiâa Merrouche, Noureddine Bouras, Yannick Coppel, Florence Mathieu, Nasserdine Sabaou, Ahmed Lebrihi
DOI: http://dx.doi.org/10.1111/j.1574-6968.2011.02246.x 41-46 First published online: 1 May 2011

Abstract

Dithiolopyrrolone antibiotics, produced by several microorganisms, are known for their strong antimicrobial activities. This class of antibiotics generated new interest after the discovery of their anticancer and antitumor properties. In this study, four new antibiotics were purified from the fermentation broth of Saccharothrix algeriensis NRRL B-24137 and characterized as dithiolopyrrolone derivatives. These new dithiolopyrrolone antibiotics were induced by adding sorbic acid, as precursor, at a concentration of 5 mM to the semi-synthetic medium. The analysis of the induced antibiotics was carried out by HPLC. The maximal production of the antibiotics PR2, PR8, PR9 and PR10 was 0.08±0.04, 0.21±0.04, 0.13±0.03 and 0.09±0.00mgL−1, respectively, obtained after 8 days of fermentation. The chemical structures of these antibiotics were determined by 1H- and 13C-nuclear magnetic resonance, mass and UV-visible data. The four new dithiolopyrrolone antibiotics – PR2, PR8, PR9 and PR10 – were characterized, respectively, as crotonyl-pyrrothine, sorbyl-pyrrothine, 2-hexonyl-pyrrothine and 2-methyl-3-pentenyl-pyrrothine. The minimum inhibitory concentrations of the new induced antibiotics were determined.

Keywords
  • new dithiolopyrrolone antibiotics
  • Saccharothrix algeriensis
  • precursors
  • sorbic acid
  • fermentation
  • antimicrobial activity

Introduction

Actinomycetes are filamentous bacteria that naturally inhabit soils. They are of great importance in biotechnological process because of their ability to produce a large number of antibiotics and other bioactive secondary metabolites. Saccharothrix algeriensis NRRL B-24137 (=DSM 44581) is an actinomycete that produces bioactive compounds belonging to the dithiolopyrrolone class of antibiotics (Lamari, 2002a,b; Zitouni, 2004). Dithiolopyrrolones are members of the pyrrothine class of naturally occurring antibiotics that contain N-acyl derivatives of 6-amino-4,5-dihydro-4-methyl-5-oxo-1,2-dithiolo[4,3-b]pyrrole. Dithiolopyrrolone derivatives were previously identified from the culture broth of certain Streptomyces spp. (Okamura, 1977; De la Fuente, 2002) and from other microorganisms such as the symbiotic bacteria Xenorhabdus spp. (McInerney, 1991) and the marine bacterium Alteromonas rava (Shiozawa, 1997).

Dithiolopyrrolone antibiotics have strong activities against a variety of Gram-positive and Gram-negative bacteria, yeasts, filamentous fungi and amoeboid parasites (Celmer & Solomons, 1955; Webster, 2002; Lamari, 2002a). Furthermore, this class of antibiotics exhibits protozoicidal, larvicidal and insecticidal activities (Šturdíková, 1990; Webster, 2002), and possess outstanding antiallergic action (Stahl, 1988). Dithiolopyrrolones also have strong activity against several human cancer cell lines and are especially useful in the treatment of malignant mammary cells (Webster, 2000; Minamiguchi, 2001).

The previous studies showed that S. algeriensis produces five dithiolopyrrolone derivatives characterized by their different N-acyl groups (R): acetyl-pyrrothine (thiolutin), iso-butyryl-pyrrothine, butanoyl-pyrrothine, senecioyl-pyrrothine and tigloyl-pyrrothine (Lamari, 2002a,b; Bouras, 2006a) (Fig. 1). Furthermore, the addition of precursors to the culture medium led to the modification in production levels of known dithiolopyrrolones (Bouras, 2006a,b) and also to precursor-directed biosynthesis of new dithiolopyrrolone analogues (Bouras, 2007, 2008). Consequently, S. algeriensis has the ability to produce a wide range of dithiolopyrrolones based on different acyl-CoA depending on the precursors added (Bouras, 2007, 2008). Recently, Merrouche (2010) showed that the addition of valeric acid at a concentration of 5mM induced the production of three new dithiolopyrrolone derivatives: formyl-pyrrothine, valeryl-pyrrothine and iso-valeryl-pyrrothine (Fig. 1).

Figure 1

Structure of dithiolopyrrolone antibiotics produced by Saccharothrix algeriensis NRRL B-24137. *Dithiolopyrrolones induced by adding valeric acid alone.

In the present work, new dithiolopyrrolone antibiotics from S. algeriensis have been induced by adding sorbic acid and subsequently purified and characterized. The minimum inhibitory concentrations (MIC) of the new induced antibiotics against several microorganisms were determined.

Materials and methods

Producing strain

Saccharothrix algeriensis NRRL B-24137 (Zitouni, 2004) was grown and maintained at 4°C on slants of International Streptomyces Project 2 (ISP 2) medium (Shirling & Gottlieb, 1966).

Fermentation studies of S. algeriensis with and without sorbic acid

Culture conditions

A mature slant culture of the strain S. algeriensis was inoculated into 500mL Erlenmeyer flasks, each containing 100mL of a basal semi-synthetic medium (SSM) consisting of 10g glucose D+ (Fisher Labosi), 2g (NH4)2SO4 (Prolabo), 2g NaCl (Fisher Labosi), 0.5g KH2PO4 (Acros), 1g K2HPO4 (Acros), 0.2g MgSO4·7H2O (Acros), 5g CaCO3 (Prolabo) and 2g yeast extract (Difco), in 1L distilled water. The pH of the medium was adjusted to 7 using a 2M NaOH solution before autoclaving. The sorbic acid (Fluka), at a concentration of 5mM, was supplied to the basal SSM prior inoculation. The inoculated cultures were put on a rotary shaker at 240r.p.m. at 30°C for 10 days.

Kinetics of antimicrobial products, growth and pH

All kinetics was assessed on SSM supplemented with sorbic acid (at 5mM) and control (without sorbic acid). The activity against Bacillus subtilis and Mucor ramannianus, used as test microorganisms, was regularly recorded each day by the agar diffusion method (well technique; each well of 10mM in diameter made in the ISP 2 agar plate was filled with 200μL of supernatant).

Dry cell weights were determined as described by Bouras (2006a) and expressed as gram per litre. The pH value was measured with a pH meter (Consort C 832, Consort, NY). All tests were repeated two times from two separate cultures.

Kinetics of antibiotic production and HPLC analysis

The culture filtrate was extracted with an equal volume of dichloromethane and the organic layer was dried with anhydrous sodium sulphate and concentrated under vacuum to generate a crude extract. The extract was concentrated to dryness under vacuum on a Rotavapor, and dissolved in 1mL of methanol as crude extract. The analysis of antibiotics induced by addition of sorbic acid in the SSM was carried out by a HPLC system equipped with a C18 reverse phase column (Uptisphere UP5ODB, 150 × 4.6mM; BioTek). The samples were analysed and quantified as described by Lamari (2002b) and Bouras (2006a).

Purification of induced antibiotics

The bacteria were cultivated in 500mL Erlenmeyer flasks, each containing 100mL of SSM supplemented with sorbic acid (5mM). For the purification, cultures were combined to obtain 15L. The mycelium was separated, and the culture broth was extracted with dichloromethane on the eighth day of fermentation. The concentrated extracts were partially purified on preparative silica gel 60 plates (Merck Art 5735, Kieselgel 60F 254) and separated by a mixture of ethyl acetate and methanol (100:15v/v). Two active bands were obtained as yellow (AJ) and yellow-orange (PS) bands at retention factor (Rf) values of 0.52 and 0.59, respectively. After elution with methanol, crude AJ and crude PS were obtained and purified by HPLC. Semi-preparative HPLC was performed on a Waters system using a C18 column (UP5ODB, 250 × 7.8mM). The samples were analysed by linear gradient elution using methanol as solvent A and ultra pure water as solvent B. The separation gradient started with 40% solvent A and 60% solvent B, and reached 100% solvent B and 0% solvent A in 30min, using a flow of 1.5mLmin−1. The detection of compounds was carried out at 390 and 220nm.

Chemical characterization of induced antibiotics

UV-visible absorption spectra of induced antibiotics were determined with a Shimadzu UV 1605 spectrophotometer. The molecular weights of the compounds were obtained by electron impact MS (EIMS) recorded at 70eV with a Nermag R-10-10C spectrometer. The nuclear magnetic resonance (NMR) sample was prepared by dissolving the pure molecules (PR2, PR8, PR9 and PR10) in 600μL of CD2Cl2. One- and two-dimensional (2D) 1H and 13C experiments were recorded on a Bruker Avance 500 spectrometer equipped with a 5mM triple resonance inverse Z-gradient probe (TBI 1H, 31P, BB). All chemical shifts for 1H and 13C are relative to tetramethylsilane (TMS) using 1H (residual) or 13C chemical shifts of the solvent as a secondary standard. The temperature was set at 298K. All the 1H and 13C signals were assigned on the basis of chemical shifts, spin–spin coupling constants, splitting patterns and signal intensities in 1H–1H COSY45, 1H–13C HMQC and 1H–13C HMBC experiments.

Antimicrobial MIC of purified antibiotics

The MIC of antibiotics were determined by a conventional agar dilution method using ISP 2 medium. The antimicrobial activity was observed after 24–48-h incubation at 37°C for bacteria and 48–72-h incubation at 28°C for fungi and yeasts.

Results

Kinetics of antimicrobial products of S. algeriensis

The results of evolution of antimicrobial products of S. algeriensis are shown in Fig. 2. The antimicrobial activity started earlier in the presence of sorbic acid (third day of fermentation against M. ramannianus and fourth day against B. subtilis) as compared with control (seventh day of fermentation against M. ramannianus and sixth day against B. subtilis). Saccharothrix algeriensis exhibited better antimicrobial activity after addition of sorbic acid compared with the control. The maximal antifungal activity (25mM diameter inhibition after 9 days of fermentation) was greater than the maximal antibacterial activity (15mM diameter of inhibition after 7 days of fermentation).

Figure 2

Effect of addition of sorbic acid to the SSM on evolution of biomass, antimicrobial activity and production of new dithiolopyrrolones by Saccharothrix algeriensis: (a) SSM without adding sorbic acid (control); (b) SSM with sorbic acid (at 5mM). ▪, Evolution of biomass; •, activity against Bacillus subtilis; ▴, activity against Mucor ramannianus; values do not include the diameter of wells (10mm). (c) Production of new obtained dithiolopyrrolones. ⋄, PR2; □, PR8; Δ, PR9; and ○, PR10.

Effect of addition of sorbic acid on pH of the medium, growth and antibiotic production in S. algeriensis

The actinomycete S. algeriensis produces five known dithiolopyrrolones (thiolutin, iso-butyryl-pyrrothine, butanoyl-pyrrothine, senecioyl-pyrrothine and tigloyl-pyrrothine) in the SSM (without precursors) as reported by Lamari (2002b). Importantly, the addition of sorbic acid to the SSM induced the production of four new dithiolopyrrolones (PR2, PR8, PR9 and PR10). The retention times of these new induced compounds (PR2, PR8, PR9 and PR10) were recorded at 28.24, 36.86, 37.16 and 37.82min, respectively. The growth of S. algeriensis was influenced by the addition of sorbic acid. In SSM broth (control), the dry cell weight reached a maximum after 5 days of fermentation (0.65±0.05gL−1) and then decreased to reach a value of 0.15±0.03gL−1 at the end of fermentation (after 10 days). However, by addition of sorbic acid, the dry cell weights reached a maximum of 1.30±0.08gL−1 (also obtained after 5 days of fermentation) and then decreased 0.32±0.06gL−1 at the end of fermentation.

Moreover, the sorbic acid allowed a high specific growth rate (μmax) of 0.074±0.004h−1, in comparison with 0.045±0.002h−1 with control. In addition, the optimal production of new dithiolopyrrolones PR2, PR8, PR9 and PR10 was observed during the idiophase and was generally dissociated from growth. The maximal production of the antibiotics PR2, PR8, PR9 and PR10 was 0.08±0.04, 0.21±0.04, 0.13±0.03 and 0.09±0.00mgL−1, respectively, recorded on the eighth day of fermentation. The production of thiolutin was reduced three times more after addition of sorbic acid (0.29±0.08mgL−1) than with the control (0.89±0.09mgL−1). Moreover, the final pH at the end of fermentation (after 10 days) was 7.92±0.06 in the presence of sorbic acid as compared with 8.16±0.04 with the control.

Purification and characterization of induced antibiotics

The culture broth with antimicrobial activity was partially purified, and the thin-layer chromatography plates showed two bands (AJ and PS). The analysis by semi-preparative reversed-phase HPLC showed that the AJ band was composed of one compound (thiolutin); however, the PS band contained eight compounds: iso-butyryl-pyrrothine, butanoyl-pyrrothine, senecioyl-pyrrothine, tigloyl-pyrrothine (Lamari, 2002a) and four induced unknown compounds. These last four compounds were purified by HPLC, and all appear yellow and exhibit antimicrobial activity. The UV-visible spectra of each of the induced compounds showed three absorption maxima. Compound PR2 absorbed at 203, 304 and 395nm, PR8 at 202, 270 and 413nm, PR9 at 204, 303 and 402nm and PR10 at 202, 304 and 398nm. The molecular weights of PR2 and PR8 are m/z 254 and 280, respectively. PR9 and PR10 have the same molecular weight (m/z 282).

Compounds PR2, PR8, PR9 and PR10 show common 1H- and 13C-NMR spectral features: two carbonyl groups (δc 167.0∼166.6 and δc 164.8∼163.8), two sp2-hybridized quaternary carbons (δc 137.4∼136.9 and δc 132.1∼131.6), one olefinic group (δH 6.71∼6.66 and δc 108.7∼108.3), one N-CH3 group (δH 3.36∼3.35 and δc 28.0∼27.4), and one NH group (δH 7.60∼7.43). These 1H and 13C signals are typical of dithiolopyrrolone derivatives. Compound PR2 showed two additional sp2 methines (δH 6.99 and 5.98 and δc 142.8 and 123.2) and one additional methyl group (δH 1.93 and δc 17.4). The 2D 1H–1H and 1H–13C experiments made it possible to confirm the presence of a 2-butenamide side chain (Fig. 3). The E-geometry of the double bond was obtained on the basis of the coupling constant of H9–H10 (15.2Hz). Compound PR8 showed four additional sp2 methines (δH 7.30, 6.27, 6.26 and 5.92 and δc 143.2, 140.0, 129.3 and 119.3) and one additional methyl group (δH 1.90 and δc 18.4). The 2D 1H–1H and 1H–13C experiments clearly revealed that PR8 contained a 2,4-hexadienamide side chain (Fig. 3). The E,E-geometry of the double bond was deduced from the coupling constant of H9–H10 (15.0Hz) and of H11–H12 (15.1Hz, obtained from simulation). Compound PR9 showed two additional sp2 methines (δH 6.98 and 5.95 and δc 147.5 and 121.9), two additional sp3 methylenes (δH 2.25 and 1.54 and δc 34.1 and 13.4) and one additional methyl group (δH 0.98 and δc 13.4). The 2D 1H–1H and 1H–13C experiments established the presence of a 2-hexenamide side chain (Fig. 3). The E-geometry of the double bond was obtained on the basis of the coupling constant of H9–H10 (15.2Hz). Compound PR10 showed one additional sp2 methine (δH 5.72 and δc 115.7), one sp3 methylene (δH 2.21 and δc 34.2) and two additional methyl groups (δH 2.24 and 1.12 and δc 19.1 and 12.1). The 2D 1H–1H and 1H–13C experiments made it possible to confirm the presence of 2-pentenamide, 3-methyl side chain (Fig. 3). The geometry of the double bond was assigned as E by the long-range 3JHC coupling constants between H9 and C11 (<2Hz) and between H9 and C13 (∼7Hz).

Figure 3

Structure of new dithiolopyrrolones PR2, PR8, PR9 and PR10 induced by adding sorbic acid.

Antimicrobial activity of purified dithiolopyrrolone antibiotics

The antimicrobial activity of the new dithiolopyrrolone antibiotics (PR2, PR8, PR9 and PR10) is shown in Table 1. The antibiotic PR8 showed higher activity than other compounds against Gram-positive bacteria. The antibiotics PR2 and PR9 were not active against Aspergillus carbonarius and the phytopathogenic fungi Fusarium oxysporum f. sp. lini, Fusarium graminearum and Fusarium moniliforme. However, the antibiotics PR8 and PR10 showed a moderate activity against all fungi and yeasts tested. None of the new induced antibiotics showed activity against Gram-negative bacteria.

View this table:
Table 1

 Antimicrobial MIC (μgmL−1) of new dithiolopyrrolone antibiotics produced by Saccharothrix algeriensis

New dithiolopyrrolones
Test organismPR2PR8PR9PR10
Bacillus subtilis (ATCC 6633)7520>10020
Bacillus coagulans (CIP 6625)7530>10020
Listeria monocytogenes (CIP 82110)40102010
Micrococcus luteus (ATCC 9314)30107575
Staphylococcus aureus (CIP 7625)3010>100100
Agrobacterium tumefaciens (no. 2410 LB)>100>100>100>100
Escherichia coli (ATCC 10536)>100>100>100>100
Klebsiella pneumoniae (CIP 82.91)>100>100>100>100
Salmonella enterica (CIP 81.3)>100>100>100>100
Pseudomonas aeruginosa (CIP A22)>100>100>100>100
Aspergillus carbonarius (M333)>10030>1002
Fusarium oxysporum f. sp. lini (Foln 3)>10075>10050
Fusarium moniliforme>10075>1005
Fusarium equiseti50207530
Fusarium culmorum50505030
Fusarium graminearum>10050>10030
Mucor ramannianus (NRRL 1829)5020405
Penicillium expansum7520>10040
Candida albicans (IPA 200)>10050502
Saccharomyces cerevisiae (ATCC 4226)30503010
  • * The test microorganisms without an accession number were from our laboratory collection.

Discussion

Dithiolopyrrolones are known to be produced by several species of Streptomyces, Xenorhabdus and Alteromonas. The actinomycete S. algeriensis produces five dithiolopyrrolones in the basic medium (without precursors): thiolutin, iso-butyryl-pyrrothine, butanoyl-pyrrothine, senecioyl-pyrrothine and tigloyl-pyrrothine (Lamari, 2002b). This actinomycete has a great ability to produce a wide range of dithiolopyrrolone derivatives that, depending on the composition of the culture medium, nature and concentration of precursors added and an enzymatic system, are involved in attaching a variety of radicals (R) into pyrrothine ring (Bouras, 2006a,b, 2007, 2008; Chorin, 2009). The data presented above show that the addition of sorbic acid at a concentration of 5mM to the SSM as a precursor has induced the production of four new peaks, as revealed by HPLC analysis. These induced compounds did not correspond to known dithiolopyrrolones with respect to retention time, but they were identified as dithiolopyrrolone derivatives by their spectral characteristics (UV spectra, EIMS and NMR).

From MS and 1H- and 13C-NMR spectroscopic analyses, as well as by comparison with all dithiolopyrrolone derivatives reported in the literature, the structures of the four new dithiolopyrrolones (PR2, PR8, PR9 and PR10) were characterized as N-acyl derivatives of 6-amino-4,5-dihydro-4-methyl-5-oxo-1,2-dithiolo[4,3-b]pyrrole. The four compounds showed a prominent fragment ion of m/z 186 and indicated by the EIMS spectrum an extra methyl group in the heterocyclic ring (corresponding to the empirical formula C6H6N2OS2) as reported for other dithiolopyrrolones (McInerney, 1991; Lamari, 2002b).

On the basis of NMR and MS data, the molecular formula of PR2 was determined as C10H10N2O2S2 (Fig. 3). The antibiotic PR8 was determined as C12H12N2O2S2, suggesting an intact direct incorporation of the sorbic acid into pyrrothine ring. The results of Bouras (2008) showed that addition of precursors into the culture medium, such as organic acids, led to precursor-directed biosynthesis of new dithiolopyrrolone analogues. In the same context, Chorin (2009) suggest that the enzymatic reaction of pyrrothine acylation takes part in the dithiolopyrrolone biosynthetic pathway in S. algeriensis, which is able to use acyl-CoA with different structures (acetyl-CoA and benzoyl-CoA) as substrates to produce corresponding dithiolopyrrolones (acetyl-pyrrothine and benzoyl-pyrrothine). Furthermore, the antibiotic PR9 showed the same molecular weight as PR10 (m/z 282) with the same molecular formula C12H14N2O2S2, suggesting isomeric compounds.

The new dithiolopyrrolones (PR2, PR8, PR9 and PR10) were named, respectively, crotonyl-pyrrothine, sorbyl-pyrrothine, 2-hexonyl-pyrrothine and 2-methyl-3-pentenyl-pyrrothine.

Our results showed that the antibacterial and antifungal activities of the newly obtained dithiolopyrrolones are related to their variable acyl groups. The antibiotic PR8 (sorbyl-pyrrothine) showed higher activity than other compounds against Gram-positive bacteria. The new dithiolopyrrolone antibiotics showed a moderate activity against all fungi and yeasts tested (except for PR2 and PR9, which are not active against A. carbonarius, F. oxysporum f. sp. lini, F. graminearum or F. moniliforme). Interestingly, the antibiotic 2-methyl-3-pentenyl-pyrrothine (PR10) showed higher activity against A. carbonarius and Candida albicans, than showed by any of the other dithiolopyrrolones produced by S. algeriensis. In fact, the biological activity of dithiolopyrrolones is strongly influenced by the nature of variable acyl groups, as reported previously (Oliva, 2001; Li, 2007; Guo, 2008). Furthermore, none of the newly obtained antibiotics showed any activity against Gram-negative bacteria; similar results have been obtained with other dithiolopyrrolones produced by S. algeriensis (Lamari, 2002a; Merrouche, 2010).

Footnotes

  • Editor: Paolina Garbeva

References

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