Folia Microbiol. 48, 563-571 (2003 )
Alternative Sources of Biological Active Substancesia
V. BĚHAL
Institute of Microbiology, Academy of Sciences of the Czech Rrepublic, 142 20 Prague,
Czechia e-mail behal@biomed .cas.cz
ABSTRACT. The occurence of resistant strains of pathogenic organisms, comming into existence of new fatal diseases, requirement of active substances against microorganisms applicable as biological weapons press scientists and companies to look for biological active substances in microorganisms which are not traditional sources of these compounds. New substances are discovered mainly in Myxobacteria, Pseudomonas, Nocardia, Basidiomycetes , marine microorganisms, Enterobacteriacea, Halobacteriacea, hyperthermophiles etc.
Majority of antibiotics and substances with diverse biological activity used in medicine are produced by actinomycetes, nonfilamentous bacteria and fungi. Other microorganisms such as myxobacteria, pseudomonads, nocardia, basidiomycetes , marine microorganisms, enterobacteria, halobacteria, hyperthermophyles etc. are investigated for new biologically active metabolites.+
INTRODUCTION
Resistance of pathogenic microorganisms against antibiotics, occurence of new fatal diseases, especially viral diseases, requirement of active substances against microorganisms applicable as biological weapons, discovery of different biological activities in compounds produced by microorganisms leads to searching of new substances in microorganisms that are not traditional sources of biological active substances or research of microorganisms living under extreme conditions. The majority of the known antibiotics are produced by actinomycetes, fungi or nonfilamentous bacteria. These microorganisms are relatively simply cultivated and using simple interferences such as UV irradiation, chemical mutation can be obtained mutants that are able to produce higher quantity of product than original strain.
Industrial production of compouns from these microorganisms is well managed. Above mentioned microorganisms are not pathogens and they are not dangerous for attending staff.
Looking for new chemical structures with interesant biological activity are researched microorganisms that are difficultly cultivated, such as Myxobacteria , pathogen microorganisms of the genus Pseudomonas a Nocardia or industrially difficult cultivated
Basidiomycetes . Besides terrestrial microorganisms are researched also their marine counterparts that live in a quite different environment and would be expected to have a different metabolic pathways and to produce compound which posses unique structure and activities. Microorganisms living under extreme conditions, in which other microorganisms can not grow, are also investigated.
MYXOBACTERIA
Myxobacteria have been regarded as a promissing source of new biological active metabolites. About 80 different basic compounds and 450 structural variants have been

characterized in myxobacteria (Reichenbach 2001). Mechanisms of action of myxobacteria products are very rare: electron transport inhibitors, substances that act on the cytoskeleton, inhibitors of nucleic acid polymerases. Also immunosuppresive cyclic peptides were isolated from myxobacteria (Vollbrecht et al . 2002). In Myxococcus stipitatus were found melithiazols having β-methoxyacrylate sceleton, for example melithiazol A (Fig. 1), which showed high antifungal activity and blocked the electron transport (Sasse et al . 1999). Inhbition of electron transport is relatively rare mechanism of action among bacterial compounds, but is often found in products produced by myxobacteria.
An antifungal metabolite, with similar βmethoxyacrylate structure-haliangicin (Fig. 2) was isolated from marine myxobacterium
(Fudou et al . 2001), which is also a respiration inhibitor. Myxobacteria Sorangium cellulosum produces antifungal antibiotic sopraphen A and cancerostatics epothilons (Gerth et al.
1996). The molecule of epothilons is synthesized by polyketosyntase type I and peptide synthase of the non-ribosomal type. Myxobacteria produce such polyketides with moiety synthesized from amino acids but also a variety of polypeptides (Bayer et al . 1999).
PSEUDOMONAS
Bacteria of the genus Pseudomonas produce a diverse array of potent antifungal metabolites. Some of them are antagonistic to several plant patogenic fungi. These include simple metabolites such as 2,4-diacetylphloroglucinol, phenazine-1-carboxylic acid and pyrolnitrin [3-chloro-4-(2´-nitro-3´-chlorophenyl)-pyrrole], as well as the complex macrocyclic lactones (Ligon et al . 2000). Pseudomonas are used for degradation of different pollutants. Some products of degradation can have antibiotic activity (Sbat et al . 2001).
Pseudomonas sp. produced also metal-containing antimycoplasma agents, zinc-containing micacocidin A, Cu-containing micacocidin B and Fe-containing micacocidin C (Fig.3 )
(Kobayashi et al.
1998a, Kobayashi et al.
1998b). The bacterium Pseudomonas aureofaciens produced compounds that were identified as the novel butyrolactones (Fig. 4) (Gamard et al.
1997). They molecule is comparable with actinomycete metabolites known as autoregulators,
A-factor from Streptomyces griseus or V irginiae butanolide C from Streptomyces virginiae
(Běhal 2000). Karalicin (Fig. 5), a compound with antiviral activity was isolated from
Pseudomonas fluorescens/putida (Lampis et al . 1996a, Lampis et al . 1996b).
NOCARDIA
Nocardia are human patogens but several new bioactive metabolites were isolated from Nocardia strains mainly from Nocardia brasiliensis.
Nemoto et al . (1997) isolated antraquinone antibiotics brasiliquinone A, B and C (Fig. 6) active against Gram-positive bacteria and multiple resistant P288/ADR tumor cells. Substances with cytotoxic activity, nocardicyclins A and B (Fig. 7) were isolated from Nocardia pseudobrasiliensis (Tanaka et al.
1997). Tubelactomicin A (Fig. 8), a product of Nocardia sp. showed strong activity against acid-fast bacteria including the drug-resistant strains (Igarashi et al . 1997). Next compound inhibited the growth of mycobacteria – nothramycin (Fig. 9) was isolated from Nocardia sp.
MJ896-43F17 (Momose et al . 1998).
New antitubercular drugs are necessary because the number of antitubercular drugs, especially against resistant strains, is less than that of other antibacterial drugs.
BASIDIOMYCETES
Bioactive metabolite from Basidiomycetes are known for long a time and they are used in traditional medicine of many cultures. In spite of fact that Basidiomycetes content a
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large scale of bioactive substance, such as immunossuppresives, cancerostatics, substances with antiviral activity etc ., compounds from Basidiomycetes produced industrially is comparatively few, obviously because of difficultes with their industrial cultivation and preparation of high-producing mutants. An interesting substance, collybial, was isolated from basidiomyces Collybia confluens, it inhibited grow of Gram-positive bacteria as well as some virusis (Simon et al. 1995). A cytotoxic and phytotoxic antibiotic, omphalone, was isolated from basidiomycetes Lentinellus omphalodes (Stark et al.
1991). Basidiomycetes produce three families of organohalogen metabolites: halomethanes, halogenated aromatics and haloaliphatic compounds. These organohalogen metabolites have demonstrable physiological roles as antibiotics, as methyl donors and as substrates for H
2
O
2
-generating oxygenases (Field et al . 1995). The group of sesquiterpenoid aromatic esters (Midoland et al . 1982, Donnelly et al . 1985) was enlarged by three compounds isolated from Armillariella melleamelleolides
(Fig. 10) (Momose et al.
2000). These compounds showed antimicrobial activities.
MARINE
Searching of substances with new biological activities and looking for substances active against resistant pathogens intented also to microorganisms living in marine or on marine plants and in marine snow. Several secondary metabolies were isolated from marine organisms too. Priority in research is founding compounds active against resistant Grampositive pathogens such as Staphylococus aureus, Enterococcus fecalis a E. feacius , which are present in hospitals. Especialy are searched compounds active against vancomycin-resistant pathogens because vancomycin is active against microorganisms resistant against other antibiotics. An attention is aimed also to “cationic peptide antibiotics”, that are widespread in nature where they play an important role in the innate immune systems protecting living organisms from microbial infection. The advantage of these peptide antibiotics is that they kill bacteria quickly, in part by physically disrupting cell membranes. A novel peptide antibiotic, active against vancomycin-resistant enterococci strains and methicililin-resistant S. aureus strains, bogorol A (Fig. 11) was isolated from marine Bacillus sp. (Basrby et al . 2001). Also sesterterpene produced from marine microorganisms aspergilloxide (Fig. 12), was isolated from fungal genus Aspergillus (Cueto et al . 2002) . Polyethers clavidol and lactodehydrothyrsiferol (Fig. 13) were isolated from sea algae Laurentia viridis (Sauto et al . 2002). These types of substances are studied because several of them have cytotoxic effect as well as antiviral inhibitory activity. There are known also enzyme inhibitors isolated from marine microorganisms. Inhibitors of xanthine oxydase - akalone and hydroxyakalone (Fig. 14) were isolated from bacterium Agrobacterium aurantiacum (Izumida et al . 1997). This enzyme participate on oxidation of hypoxanthine into uric acid in the blood. Thiocoraline (Fig.15), a potent antitumor compound, as well as substances with strong antimicrobial activity against
Gram-positive microorganisms, was isolated from marine Micromonospora (Romero et al .
1997). Thiocoraline does not inhibit topoisomerase I and II, but inhibits RNA synthesis. It is a depsipeptide with several sulphur atoms in its molecule. Amagata et al . (1998) searching for new antitumor substances isolated from a strain Penicillium waksmanii present on brown alga
Sargassum ringgolianum new cytotoxic metabolites pyrenomycin D and E (Fig.16)..
ENTEROBACTERIACEA
Several members of Enterobacteriacea produce substances that inhibit phylogenetically related species. Their biological active products are named colicins or microcins. These substances are proteins ranging size from 0.5 kDa to 80 kDa. Substances with smaller molecule (less than 10 kDa) are called microcins (Moreno et al . 1995). Peptides
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of microcins are synthesized on ribosomes and modified posttraslationally. Peptides of microcins contain combinations of thioether bridges, alpha, beta-unsaturated amino acids, Damino acids, N-, C-terminal and heteroaromatic backbone modifications, all of which arise from posttranslational modification of Ser, Thr, Cys and Gly residues in a ribosomallysynthesized precursor polypeptide (Kaiser at al . 1998). A complex system of plasmid and chromosomal genes is involved in the production and resistance to microcins. (Khmel 1999).
Because microcins are active against related species, very important is the host cell resistence
(imunity) to the product. Microcins are produced during stationary phase growth and they are secreted into the extracellular medium by active and dedicated mechanisms. Larger colicins are passively released through the selective leakage that follows activation of a cellular phospholipase by lysis protein (Moreno et al . 1995).
Similar bioactive polypeptides, produced by Gram-positive bacteria are bacteriocins and their subgroup lantibiotics that possess extensive post-translational modifications (Gasson
1995). Some of them are known for a long time. Nisin was discovered as a product of
Lactococus lactis and subtilin is produced by Bacillus subtilis ATS6633. Next lantibiotics were isolated from Staphylococcus gallinarum, Staphylococcus epidermis, Lactobacillus sake etc . Over 30 lantibiotics are known presently. Some lantibiotics have been studied for use in treating skin infection, some of them have antiviral activity. The small size and surface activity of these peptides also may allow them to enhance the transport of therapeutic compounds across cell membranes (Bower et al.
2001). Lantibiotics funtion during disruption of the membrane of their targets (Garneau et al.
2002). The main problem for broad application of peptide antibiotics in clinical praxis is that they are difficult to obtain in high quantity. Methods of isolation using ionic membranes may enable earn sufficient quantity of drags (Recio et al . 2000).
HALOBACTERIACEA
The Halobacteriacea are extremely halophilic archaeobacteria living in aquatic hypersaline environments. Production of archaeal proteinaceus antimicrobials-halocins from halophiles is nearly universal feature of the rod-shaped haloarchaea. They are externalized into the environment, where they kill or inhibit other haloarchaeons (Price and Shand 2000).
Halocins are ribosomally synthesized proteins or peptide antimicrobials (O´Conors and Shand
2002), they are diverse in size, consisting of proteins as large as 35 kDa and peptide
“microhalocins” as small as 3.6 kDa. Microhalocins are hydrophobic rober drags, withstanding heat, desalting and expose to organic solvents. Mechanisms of action of halocins is known only for halocin H6 which disrupts the haloarchaeal Na+/H+ antimporter (Meseguer et al . 1995). The discovery that halocin H7 inactivates the same target in mammals as it does in halobacteria suggests the possibility that other halocins have also clinical applications
(O´Coners and Shand 2002).
HYPERTHERMOPHILES
Microorganisms living in thermal springs were isolated and investigated for presence of biologialy active substances. Strains of genus Bacillus isolated from thermal springs of the
Kamchatka Peninsula produce very stable peptide antibiotics active against Gram-positive bacteria. They survived boiling for 30 min, were resistant to trypsin and chymotrypsin and they were stable at a pH range of 2.0-9.0 (Esikova et al . 2002). Biological active substances produce also thermophillic actinomycetes (Edvards 1993).
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CONCLUSIONS
Research of different types of microorganisms proved that majority of them produce some biological active substances. They have variegated structures and their biological activities are diverse. Biological active compounds found in Enterobacteriacea and
Halobacteriacea were peptides synthesized on ribosomes. Biological active substances from others microorganisms, as show the figures presented in this review, pertain to different types of secondary metabolites as we know in traditional secondary metabolites producers.
Myxobacteria often produce compounds that are synthesized both by polyketide synthasis and by polypeptidases (epothilons) (Běhal 2003).
A reason why microorganisms synthesize secondary metabolites is still unclear. Some of them, especially peptides, serves as signal molecules. Specific biological activity is matter of chance. Compounds with similar structure can have different biological activity. If antibiotics serve for competition to other microorganisms in ecosystem is discutabil,. production of secondary metabolites in nature is low so that other microorganisms can acquire quickly resistence. The substance with antibiotic activity is usually only one from many other secondary metabolites that are produced by the microorganisms.
A searching of new biological substances is very expensive mater, especially looking for antibiotics active against viruses but occurrence of new fatal diseases forces this research.
This work was supported by the Grant Agency of the Czech Republic (grant no. 204/01/1004).
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