The distribution and specificity of extracellular nucleases produced by marine fungi belonging to eleven genera, namely: Alternaria, Aspergillus, Aureobasidium, Chaetomium, Fusarium, Gliomastix, Humicola, Penicillium, Scopulariopsis, Wardomyces, Periconia, have implied its important function in the organic phosphorus and nitrogen circle in the Ocean. The fungal nucleases of 64 isolates tested were more or less specific for single-stranded DNA with a high preferential specificity towards poly-U substrate with forming of 5’-phosphate mononucleotides. A couple of the nucleases were capable of RNA digesting. The highest level of extracellular nucleolytic ability was observed in Penicillium spp. isolates. The tight correlation found between extracellular nuclease activity and the rate of thymidine uptake by actively growing and sporulating marine fungus Penicillium melinii suggests that this nuclease is required for fulfilling the nucleotide pool of precursors of DNA biosynthesis during transformation of hyphae into the aerial mycelium and conidia in stressful environmental conditions.
The main portion of the organic carbon, phosphorus and nitrogen is a direct result of enzymatic processing of dissolved macromolecules of DNA, RNA and proteins in aquatic environments due to their wide occurrence [1,2]. Aquatic microorganism populations utilize nucleic acids, enabling efficiently scavenging of organic matter from the dissolved pool, rapidly cycling the nucleic acids [3, 4]. Therefore, there is considerable interest in molecular utilization mechanism of nucleic acids in marine ecosystem. Some fields of fungal community studies have been described [
It is known that a large group of fungi and filamentous bacteria generate and disseminate spores for implement of survival program in stressful environmental conditions. These events call forth increase of nuclease activity dramatically that coincides with lysis of the substrate hyphae and formation of aerial mycelium [
Only a few species of terrestrial fungi producing extracellular nucleolytic enzymes have been reported [11- 13]. No information is available on the genus or species specificity of nucleolytic enzymes in marine fungi. However, the previous data have shown the substantial difference between the marine fungi and their terrestrial counterparts [14,15]. Moreover, most of marine fungi were found to be facultative with the potential for longdistance dispersal that might enable unusually rapid propagation of epidemics in marine systems [
The purpose of the present study was to evaluate extracellular nucleolytic ability in marine fungi using different natural and synthetic substrates and its possible biological and ecological role in marine environment.
The fungal strains were isolated from marine animals and sea bottom sediments collected in the shore water of the Kuril Islands and Primorskyi krai (
maintained on sterile seawater with Wort agar [
Extracellular nucleolytic enzyme activity was determined in the fungal culture during the cells growth. For extracellular nucleolytic enzyme activity detection, cell-free supernatant after culture filtration was inoculated onto the agar plate. The agar plates were prepared with 3 g of liquefied agarose (“Hemapol”), 200 ml of 0.03 M sodium acetate buffer, pH 4.6, containing 1 mM ZnSO4 and 0.05 M NaCl, and 2 ml heat-denatured DNA (ssDNA) at concentration of 1 - 2 mg/ml. The resulting mixture was immediately poured onto marked horizontal plates with the final volume about 20 ml (8 × 12 cm and 2 mm thick). Wells of 3 mm in diameter were cut in the agar plates. Each of two-microliter samples of fungal culture in equal protein concentrations was inoculated into the wells. After incubation at 37˚C for 20 min, the result of the nuclease activity was observed at λ260. The more active fungal isolates were selected by comparison of the dark circle zones produced by enzymatic cleavage of the substrate for the further quantitative analysis.
Nuclease specificity was evaluated in a mixture of 0.5 ml 0.03 M sodium acetate buffer, pH 4.6, containing 1 mM ZnSO4 and 0.05 M NaCl, substrate in concentration of 10 OE260 and 0.02 ml of filtered fungal culture sample. The reaction mixture was incubated at 37˚C for 20 min and terminated by adding 2.0 ml of 0.5 M HCLO4 and 0.1% La(NO3)3. The mixture was centrifuged at 2000 g for 10 min. The absorbance of supernatant at λ260 was measured. One unit of the enzyme was defined as the amount that catalyzes the production of acid-soluble nucleotides increased by 1.0 absorbance unit for 20 min. Protein concentration was defined by Bredford method [
Penicillium melinii isolates were examined for the ability to take up [H3]thymidine during cell growth. [H3]Thymidine uptake was determined as a total cell-associated radioactivity by adding 1-ml samples to tubes containing 1 ml of 2 µCi/ml [H3]-labeled thymidine in nutrient-free artificial seawater. Subsamples were filtered within 5 min onto 0.2-mm-pore-diameter GFC filters (Whatman) and were washed with 5 ml of 5% trichloroacetic acid with 1% sodium pyrophosphate and ethanol. Radioactivity in the samples was measured by liquid scintillation counting (Mark III). The conidia were direct counted immediately and again after every two days of incubation. Counting of conidia was carried out as described by Bilai [
Extracellular nucleolytic ability, [H3]thymidine uptake and conidia productivity of fungi were evaluated as the average of three independent experiments. Statistical significance was analyzed by the Student’s t-test, the critical level of significance was 5%.
Recombinant DNA technique was performed using Ins T/Aclonetm PCR Product Cloning Kit, restriction endonucleases, T4 DNA ligase, Long PCR mix (Fermentas), Smart Taq Polymerase (Topotili), automatic amplifier (Eppendorf). Two pair of primers (For-1 and Rev, sequences: 5’-CAYTTYATHGGNGAYATGAC-3’ and 5’- GCCCANCKNGTNGCNGT-3’, respectively; For-2 and Rev, sequences: 5’-GAYTGGGAYACNTAYATGCC-3’ and 5’-GCCCANCKNGTNGCNGT-3’, respectively) were constructed on the base of S1-like nuclease homologue sequence alignments to amplify the active site region of S1 type nuclease using chromosomal DNA of P. melinii, P. patulum and P. chrysogenum as templates. Nand C-termini region coding sequences were determined using two pair of primers Nuc-for1 and Nuc-rev, sequences: 5’-ATGGTCTCTCTATCCAAGATTG-3’ and 5’-ATGGCTATGCTCACGTCCTTCTGAGG-3’, respectively. PCR products were cloned and sequenced using the automated PE/ABI 310 DNA sequencer and PE/ABIABI PRISM BigDye Terminator cycle sequencing Ready Reaction Kit (PE Applied Biosystems). E. coli strains TOP10 or XL1 (Evrogen) were used for standard cloning procedures. Nucleotide and amino acid sequence analysis were performed with Chromas, GenRunner, SMART programs. Nucleotide and amino acid sequences homology and similarity searches and alignments were carried out by using BLAST and ClustalW, МОЕ 9.10 facilities.
Fifty percent from more than 60 isolates tested was found to produce a different level of extracellular nuclease activity catalyzing the cleavage of ssDNA at pH 4.6 (
The marine fungi were found to produce non-specific extracellular ssDNA/RNA nucleases especially those, which was been habitable under the deep-sea conditions (
As shown in
pected, the amount of green conidia was highest at 11 days of incubation (
The structural and biochemical details of some ssDNApreferential nucleases of class 3.1.30.1 isolated from the terrestrial sources are known [13,22,23]. We have cloned and analyzed ssDNA-preferential highly active extracellular nucleases from marine fungi, namely: P. melinii (GU331890), P. patulum (formerly P. griseofulvum) (GU331891), and P. chrysogenum (GU33182).
The presence of putative signal peptide indicates that these proteins are destined for secretion to the periplasm or excretion into the environment. Gene Bank databases searching by BLAST software disclosed a significant sequence homology of these enzymes with terrestrial P. citrinum (70% identity), A. oyzae (52% identity), and N. crassa (47% identity) S1 type nucleases that suggested the same mechanism of action (
Our investigation demonstrated a large number of the fungal strains collected from the sea deepwater sources, producing highly active extracellular nucleases into marine environment. Extracellular nucleolytic ability was detected in the strains of the genera Aspergillus, Chaetomium, Fusarium, Humicola, Penicillium, Scopulariopsis that were found prolifically in the sea bottom sediments and marine animals inhabiting of the shelf waters of Japan Sea. The species of the genera Penicillium, Aspergillus Chaetomium, Fusarium were of more frequent occurrence (
Of 64 isolates tested, two were capable of RNA digesting (
some marine fungi species have a potential to safe genetic information from attacking carrier of viruses resides providing participation in host defense [
Actively growing marine fungus P. melinii (formerly P. estinogenum) collected from colonial Ascidium spp. at the depth of 270 m was examined for [H3]thymidine uptake into the cells (
the certain conditions of cultivation are required for the continuous synthesis of the enzyme (unpublished results). The aerification deficiency appears to provide the active conidia formation.
The extacellular nucleolytic ability in marine fungi, as demonstrated in this study, indicated that their enzymes, probably, are the major deliver of the nucleotide matter to the symbiothrophic organisms in the condition of the organic matter deficient (
In summary, we have shown that the marine fungi possess important extracellular nucleolytic enzymes, which are required for fulfilling the nucleotide pool of precursors of DNA biosynthesis and maintaining ecological balance. Their distribution in the deep-sea water and unique specificity confirmed the important function in the organic phosphorus and nitrogen circle in the Ocean. The potential of extracellular nucleolytic ability in marine fungi to estimate the marine environmental conditions as well as the biotechnology potential of the marine fungi nucleases will attract much attention.
Financial support was provided by grants from RFBR 08-08-00975-a and the RAS Presidium under the project “Molecular and Cellular Biology” 09-I-P22-05.