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Actinomycetes, Vol. 7, Part 3, 1996 pp.79-83 HEMAGGLUTINATING ACTIVITY OF STREPTOMYCETES FROM NATURAL AND HEAVY METAL POLLUTED SOILS V. E. KOZYRITSKAYA, H. V. VALAGUROVA, A. A. PINDRUS and K. I. ANDREYUK Institute of Microbiology and Virology, Kyiv, Ukraine
Code Number:AC96011
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ABSTRACT. Ability to produce extracellular lectins is widespread among soil streptomycetes. More than 50% of culture collection strains as well as of fresh soil isolates showed hemagglutinating activity. Active streptomycetes are more frequent in soils characterised by heavy metal pollution.
The capacity of streptomycetes to produce of lectins - substances that play an important role in carbohydrate-protein recognition - is poorly studied and so far only a few strains have been investigated (Lynevitch, 1979; Kameyama & Oishi, 1983; Lakhtin, 1987).
The aim of the present study was to investigate lectin formation by streptomycetes from a culture collection and fresh soil isolates.
MATERIALS and METHODS
Organisms. Eighty Streptomyces strains, belonging to thirty-nine species, isolated more than 20 years ago from both untilled and cultivated soils and maintained as lyophilised cultures at the Ukrainian National Collection of Microorganisms were investigated.
In addition 505 Streptomyces isolates were obtained in 1993 from a grey podzolic soil and from the same source experimentally polluted with heavy metals (nitrate salts). Amounts (mg/kg of soil) of metals were as follows: Cu (7.1 and 14.2), Cd (6.8 and 13.6), Pb (5.3 and 10.6), Sr (10.1 and 20.2), Hg (4.5 and 9.0).
Growth conditions. Strains were grown in shaken culture at 28-30 C on Czapek,s liquid medium with glucose (2.0%) and CaCO3 (0.2%) for 4 dd.
Hemagglutinating activity (HAA). HAA was determined by the hemagglutination reaction (HAR) using the method of double dilution in polystirol plates (Lucik et al., 1980). Rabbit erythrocytes were treated by trypsin and fixed by glutaraldehyde. HAA was estimate by the last dilution where HAR could still be observed and 1 Unit HAA was taken as the titre^-1 of the HAR.
RESULTS and DISCUSSION
Half of the Streptomyces collection cultures (41 out of 80) produced lectins, with a HAA ranging from 2 to 64 Units (Table 1).
Table 1. Streptomycetes from the Ukrainian National Collection of Microorganisms producing lectins and HAA Units
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Strains
Section Series Species Active/Tested HAA Units
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Azureus Coerulescens S.afghaniensis 1/2 4
S.coeliatus 1/2 8
S.coeruleoflavus 1/1 8
Glaucescens S.glaucus 3/3 4-8
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Cinereus Achromogenes S.pseudogriseolus 1/1 16
Aureus S.flaveolus 1/2 4
S.fulvostreptomycini 1/1 32
S.olivaceus 1/4 2
Violaceus S.cyanogenus 1/4 2
S.violaceoruber 1/4 2
Chrysomallus S.olivaceoviridis 0/1
S.olivoviridis 0/1
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Roseus Lavendulae- S.filamentosus 1/1 64
Roseus S.lavendosporus 1/1 64
S.lincolnensis 0/2
S.roseus 1/2 2
S.toxytricini 1/1 8
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Helvolo- Helvolus S.alboflaveolus 1/1 64
Flavus S.chrysomallus 1/1 16
S.globisporus 2/5 8
S.griseus 4/4 2-8
S.odorifer 2/2 16
S.oligocarbophilus 1/1 64
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Albus Albus S.albus 3/5 4-32
S.sindenensis 1/1 16
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S.filamentosus 2104 and S.lavendosporus 2114 (Lavendulae- Roseus series; Roseus section) (Gause et al., 1983), S.alboflaveolus 2073 and S.oligocarbophilus 2125 (Helvolus series; Helvolo-Flavus section) showed maximum activity (64 Units). A lower activity (32 Units) was observed in S.albus 2018 (Albus series; Albus section), S.fulvostreptomycini 2079 (Aureus series; Cinereus section) and in S.pseudogriseolus 2058 (Achromogenes series; Cinereus section).
As shown in Table 2, no relationship could be observed between HAA and the taxonomic status of the cultures. Although for the present study the most largely represented species in the Collection were chosen, it was not possible to ascertain any HAA specificity. In addition no correlation was found with morpho-cultural characteristics of the strains (sporophore morphology, spore surface, pigmentation).
Table 2. Morpho-cultural characteristics of streptomycetes from the Ukrainian National Collection of Microorganisms and HAA Units (+: present; -: absent; S: Spirales; RF: Recti-Flexibiles; H: hairy; SM: smooth; SP: spiny)
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Sporoph. Spore Pigment HAA
Strain Morphol. Surface Melanoid Other Units
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S.glaucus 2047 S H - + 8
S.glaucus 2118 S H - + 4
S.glaucus 2119 S H - + 8
S.griseocastaneus 2101 S SP + + 0
S.griseocastaneus 2102 S SP + + 0
S.flaveolus 2110 S H - + 0
S.flaveolus 2008 S H - + 4
S.lavendulae 2006 RF SM + - 8
S.lavendulae 2031 RF SM + - 0
S.globisporus 2048 RF SM - + 0
S.globisporus 2049 RF SM - + 0
S.globisporus 2050 RF SM - + 8
S.globisporus 2097 RF SM - + 8
S.albus 2016 S SM - - 8
S.albus 2018 S SM - - 32
S.albus 2019 S SM - - 4
S.albus 2020 S SM - - 0
S.albus 2021 S SM - - 0
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The newly isolated streptomycetes were representatives of 14 series and 4 sections (Cinereus, Albus, Helvolo-flavus and Roseus) according to Gause et al. (1983). More than half of the strains (267) were capable of forming lectins, as shown in Table 3. Cultures with high HAA (64-256 Units) were found in the Aureus, Chrysomallus and Chromogenes series (Cinereus section). Active strains however could be found also in other series.
Table 3. Lectin production by fresh soil isolates
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Section Series Number of Isolates % Lectin Producers
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Cinereus Achromogenes 82 62.2
Chromogenes 98 59.2
Violaceus 8 100.0
Aureus 20 60.0
Chrysomallus 139 77.7
Albus Albus 13 69.2
Albocoloratus 89 49.4
Helvolo-Flavus Flavus 4 50.0
Helvolus 20 75.0
Roseus Lavendulae-Roseus 11 45.5
Fuscus 17 64.7
Ruber 4 75.0
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Experimental pollution with heavy metal salts appeared to positively influence the number of lectin producers (Table 4). Natural soil showed the lowest proportion of active cultures and these in turn were characterised by low activity. High percentages of active strains were obtained with Cu, Pb, Hg and Sr and high HAA was noted in soils polluted with Hg, Sr and Cd, notwithstanding the fact that these metals can influence negatively the soil streptomycete community as a whole (Valagurova et al., 1996). Table 4. Lectin production by streptomycetes isolated from natural soil and from soil experimentally treated with heavy metal salts
------------------------------------------------------------------------ Type of Soil Number of Isolates % Lectin Producers HAA Units ------------------------------------------------------------------------ Control (Natural Soil) 49 51.02 2-16 Soil + Cu 70 84.28 2-32 Soil + Sr 134 59.70 4-256 Soil + Cd 73 53.42 4-128 Soil + Hg 52 61.54 8-256 Soil + Pb 93 65.59 2-32 ------------------------------------------------------------------------ Results obtained confirm the widespread capacity for lectin production among soil streptomycetes. Some 50% of the strains tested, both maintained for over 20 years in a culture collection and freshly isolated, showed activity.
In spite of the negative effect of heavy metal salts on microorganisms, a positive influence on lectin production has been observed. While it is known that lectins play a role in symbiosis and parasitism, their significance for free living microorganisms, such as soil streptomycetes, is not clear. In our opinion it is possible that in soil lectins could have a protective effect against heavy metal pollution.
REFERENCES Gause, G.F., T.P.Preobrazhenskaya, M.A.Sveshnikkova, L.P.Terekhova & T.S.Maksimova (1983). Key to the Actinomycetes. Nauka Publ., Moscow, 246 pp. (in Russian)
Kameyama, T. & K.Oishi (1983). Microbial lectins. Prot. Nucl. Ac. Enzym., 28: 132-145
Lakhtin, V.M. (1987). Lectins in Proteins and Carbohydrates Investigation. Results of Science and Technique. Ser. Biotechnology: 289 pp. (in Russian)
Lucik, M.D., E.N.Panasjuk & B.A.Antonjuk (1980). The Methods of Lectins (Phytohemagglutinins) Findings and Definition of Their Immunochemical Specificity. High School Press, Lvov. 20 pp. (in Russian)
Lynevitch, L.I. (1979). Lectins and carbohydrate-protein recognition at the various level of the life organisation. Adv. Biochem., 20: 71-20 (in Russian)
Valagurova, H.V., G.A.Iutinskaya, V.E.Kozyritskaya, N.I.Ivanova & K.I.Andreyuk (1996). Heavy metals effect on the streptomycete association of grey podzolic soil. Microbiol. Zh., 58 (2): 16-22 (In Ukrainian). Copyright 1996 C.E.T.A., The International Centre for Theoretical and Applied Ecology, Gorizia |
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