Antifungal peptidic compound from the deep-sea bacterium Aneurinibacillus sp. YR247

Aneurinibacillus: sp. YR247 was newly isolated from the deep-sea sediment inside the Calyptogena community at a depth of 1171 m in Sagami Bay. The strain exhibited antifungal activity against the filamentous fungus Aspergillus brasiliensis NBRC9455. A crude extract prepared from the YR247 cells by ethanol extraction exhibited broad antimicrobial activities. The antifungal compound is stable at 4–70 °C and pH 2.0–12.0. After treatment with proteinase K, the antifungal activity was not detected, indicating that the antifungal compound of strain YR247 is a peptidic compound. Electrospray ionization mass spectrometry of the purified antifungal compound indicated that the peptidic compound has an average molecular weight of 1167.9. The molecular weight of the antifungal compound from strain YR247 is different from those of antimicrobial peptides produced by the related Aneurinibacillus and Bacillus bacteria. The antifungal peptidic compound from the deep-sea bacterium Aneurinibacillus sp. YR247 may be useful as a biocontrol agent.

73 Page 2 of 8 exhibit antimicrobial and antifungal activities. Although gramicidin S has an antibiotic activity against a broad spectrum of Gram-negative and Gram-positive bacteria and several pathogenic fungi, hemolytic activity of gramicidin S restricts its use to topical applications (Mogi and Kita 2009). Under these circumstances, Bacillus species and the related bacteria have attracted much attention in the exploration for new antifungal and antibacterial substances.
The exploration of deep-sea environments has emerged as a new frontier in pharmaceutical development (Skropeta 2008). To investigate the utilities of deep-sea microorganisms, we have isolated and characterized several deep-sea microorganisms (Aono et al. 2010;Kurata et al. 2007). Cold seep areas have been found in subduction zones of continental margins, mud volcanoes, and deepsea trenches (Kulm et al. 1986;Li et al. 1999a, b), and the microbial diversity in cold-seep biological communities has received attention because of those specific microbial processes (Heijs et al. 2007;Sibuet and Olu 1998). The biological communities in the cold seep areas consist of mussels, clams, and tubeworms, and are supported by microbial chemosynthesis utilizing methane, sulphur, sulphide, and so on, seeping through the geological processes of the seafloor (Boetius et al. 2000;Fujiwara et al. 2001;Li and Kato 1999). Study of the microbial diversity of the Calyptogena sediment (depth; 1168 m) and bacterial mat (depth; 1174 m) in Sagami Bay revealed that the microbial community at this site is distinct from other cold seep sites (Fang et al. 2006). Epsilonproteobacteria, which are abundant in other cold seep sites and hydrothermal vents (Urakawa et al. 1999(Urakawa et al. , 2000, are absent from the cold-seep area of Sagami Bay. The Calyptogena microbial community in the cold seep area of Sagami Bay consists mainly of bacteria that be closely related to Shewanella sp. (Gammaproteobacteria) and Desulfobulbus sp. (Deltaproteobacteria) and some archaea that be closely related to Sulfolobus sp. (Crenarchaeota) and Methanococcus sp. (Euryarchaeota). Although a variety of bacteria and archaea have been generally elucidated in those cold seep biological communities, very little is known concerning microorganisms that exhibit antimicrobial activities.
In this study using a deep-sea sediment (depth; 1171 m) of the Calyptogena community of Sagami Bay as the isolation source, we explored the microorganism that produces an antifungal substance against filamentous A. brasiliensis. The bacterial isolate was phylogenetically characterized, and its inhibitory activity against fungus, yeast, and Gram-positive and Gram-negative bacteria was evaluated. Additionally, the properties of the antifungal compound were analyzed.

Isolation of the deep-sea bacterium with antifungal activity
A deep-sea sediment inside the Calyptogena community was collected using Hyper-Dolphin remotely operated vehicle in the Sagami Bay (35°0.072′N 139°13.477′E), Japan, at a depth of 1171 m in January 2010 (Hiyoshi et al. 2011). The deep-sea sediment was collected and immediately stored at −80 °C. After 7 months, bacterial isolation was carried out. The deep-sea sediment is fine-grained mud and an aliquot of the sample was spread on a modified LB plate consisting of (w/v) 0.1% Bacto tryptone (Difco), 0.5% Bacto yeast extract (Difco), 0.5% glucose, 1.0% NaCl, 0.2% MgSO 4 ·7H 2 O, 0.02% K 2 HPO 4 , and 1.5% agar (pH 7.4). After incubation at 37 °C for 2-3 days, several colonies had grown on the plate. For detection of the antifungal activity against the filamentous fungus A. brasiliensis NBRC9455, the colonies were inoculated onto a spore-mixed LB modified plate (pH 7.4) consisting 1.0 × 10 4 spores/ml A. brasiliensis NBRC9455 spores. After incubation at 37 °C for 3 days, colonies with inhibition zones of A. brasiliensis NBRC9455 were isolated as antifungal substance-producing microorganisms. The colonies were repeatedly purified and separated, suspended in a 15% glycerol, and stored at −80 °C.

Phylogenetic analysis of bacterial isolate
The bacterial isolate YR247, which exhibits high antifungal activity against (A) brasiliensis NBRC9455, was evaluated. To determine phylogenetic relationships, 1509 bp of the 16S rRNA gene fragment was amplified from DNA fraction of strain YR247 by PCR. DNA fraction was extracted from bacterial cell pellet of strain YR247 using an Illustra bacteria genomic Prep Mini Spin Kit (GE Healthcare, Munich, Germany). The genomic DNA of strain YR247 was used as a template. The 16S ribosomal DNA sequence was amplified and sequenced using universal primers 27f: 5′-GAG TTT GAT CCT GGC TCA G-3′ and 1525r: 5′-AAA GGA GGT GAT CCA GCC -3′ (Lane 1991). The reaction solution was 50 µl, including 0.5 µl Blend Taq (2.5 U/ µl, Toyobo Co. Ltd., Osaka, Japan), 5 µl 10× PCR Buffer for Blend Taq, 5 µl dNTPs (2 mM), 20 ng template DNA, 1 µl 27 f primer (10 µM), 1 µl 1525r primer (10 µM) and 36.5 µl ddH 2 O. The amplification was conducted by polymerase chain reaction in a PCR thermal cycler (MyCycler, Bio-Rad Laboratories Inc., Richmond, CA). The program for the PCR was as follows: denaturation at 94 °C for 7 min followed by 30 cycles of denaturation at 94 °C for 45 s, annealing at 55 °C for 45 s, and extension at 72 °C for 45 s. The DNA sequencing was carried out by FASMAC Page 3 of 8 73 DNA Sequencing Services (Kanagawa, Japan). Nucleotide substitution rates (Knuc) were determined, and a distance matrix tree was constructed by using the neighborjoining method with the CLUSTAL_X program (Kimura 1980;Saitou and Nei 1987;Thompson et al. 1997). Alignment gaps and unidentified base positions were not taken into consideration for the calculations. The topology of the phylogenetic tree was evaluated by performing a bootstrap analysis with 1000 replications (Felsenstein 1985). NJ plot software (http://pbil.univ-lyon1.fr/software/njplot. html) was used to prepare a graphical view of the phylogenetic tree. The 16S rRNA gene sequence of (B) subtilis IAM12118 T (AB042061) was used as an out group.

Production and purification of the antifungal compound
Strain YR247 was aerobically cultured in 2.4 l of LB modified media containing 0.1% Bacto tryptone (Difco), 0.5% Bacto yeast extract (Difco), 0.5% glucose, 1.0% NaCl, 0.2% MgSO 4 ·7H 2 O, and 0.02% K 2 HPO 4 (pH 7.4) at 37 °C for 24 h. After cultivation, a 13.5 g cell pellet was obtained by the centrifugation at 6000×g at 25 °C for 10 min. The crude extract was prepared according to the reference (Nakai et al. 2005). The cell pellet was resuspended in 225 ml of ethanol, and the cell suspension was then heated at 95 °C for 3.0 min. After centrifugation of the heated cell suspension at 6000×g at 25 °C for 10 min, 220 ml of the supernatant containing the antifungal compound was collected and placed on a rotary evaporator. The solvent was removed at 37 °C and 30 hPa for 30 min, and 150 mg of the residue obtained was resuspended in methanol and filtered through a 0.2 µM filter to prepare the 39 mg/ml crude extract.
The solvent of the 39 mg/ml crude extract containing the residue (150 mg) was changed to ethyl acetate. Then, the crude extract in ethyl acetate was submitted to silica gel chromatography (C200, Wako Pure Chemical, Osaka, Japan, 1.2 × 20 cm). Elution was performed using ethyl acetate, ethyl acetate:2-propanol (1:1, v/v), and methanol consecutively. Using agar-spot assay and ninhydrin coloration, active fractions were determined and collected. The residue in the active fractions was obtained by rotary evaporation at 37 °C and 30 hPa for 30 min. The residue (30 mg) was dissolved in ethanol:2-propanol (1:1, v/v) and subjected to silica gel chromatography (C200, Wako Pure Chemical, Osaka, Japan, 1.2 × 20 cm). Elution was performed using ethanol:2-propanol (1:1, v/v), and the active fraction was collected. The residue (15 mg) was concentrated from the active fraction by the rotary evaporation and resuspended in methanol. The methanol fraction was subjected to preparative thin-layer chromatography. The residue in the methanol fraction was developed on thin-layer chromatography (TLC) silica gel plates (TLC aluminum sheets, 8 × 8 cm, SilicaGel 60F254, Merck Co. USA), followed by detection of the antifungal compound by TLC bioautography overlay assay and ninhydrin reaction. The active spot area of another developed TLC plate was collected, and the antifungal compound was extracted with an aliquot of methanol. The methanol fraction containing the residue (2.3 mg) was used as the purified antifungal compound.

Detection of antimicrobial activity
Antifungal activity against A. brasiliensis NBRC9455 was determined by agar-spot and TLC bioautography overlay assays. For the agar-spot assay, 50 µl of the crude extract at 5.0 mg/ml-methanol from strain YR247 was spotted onto the surface of the spore-mixed LB modified plate (pH 7.4) consisting of 1.0 × 10 4 spores/ml A. brasiliensis NBRC9455 spores. After incubation for 3 days at 37 °C, the inhibition zone (mm) at the spotted site was measured. For the TLC bioautography overlay assay, 10 µl of the purified antifungal compound at 1.0 mg/ml was developed using acetone:distilled water (7:1, v/v) on a TLC silica gel plate (TLC aluminum sheets, 8 × 8 cm, SilicaGel 60F254, Merck Co. USA). The developed TLC plate was placed in a petri dish and covered with the spore-mixed LB modified plate (pH 7.4) containing 1.0 × 10 4 spores/ml A. brasiliensis NBRC9455 spores. After incubation for 3 days at 37 °C, the inhibition zone of the developed plate was detected. Using another developed TLC plate, the antifungal compound was detected by ninhydrin reaction. Antimicrobial activities against C. albicans NBRC1594, S. aureus NBRC12732 T , and E. coli NBRC3301 T were evaluated by the agar-spot assay described above. The crude extract (50 µl at 5 mg/ml-methanol) was spotted on each plate containing 1.0 × 10 7 cfu/ml of each microorganism cells. After incubation 1-2 days at 28-37 °C, the inhibition zone on the spotted place was checked. All experiments for evaluation of the antimicrobial activity were carried out in triplicate.

Effects of temperature, pH, and proteinase K on antifungal activity
To examine the effect of temperature, the crude extract (5 mg/ml) was incubated at 4, 10, 28, 37, 60, 70, 80, 90, and 100 °C for 6 h respectively. After the sample was cooled to room temperature, the residual antifungal activity of the treated sample (50 µl) was tested by agar-spot assay using A. brasiliensis NBRC9455.
To evaluate the pH stability of the purified antifungal compound, pH values of the crude extract (5.0 mg/ml) were adjusted from 2.0 to 12.0 at intervals of 2.0 by substitution of each solvent by ultrafiltration, and the adjusted crude extract kept at 4 °C for 12 h. Afterward, the pH values were readjusted to 7.0, and the residual antifungal activity of the 73 Page 4 of 8 treated sample (50 µl at the final concentration of 5.0 mg/ ml-distilled water) was tested by the agar-spot assay using A. brasiliensis NBRC9455.
For evaluation of the effect of proteinase K, the crude extract (5.0 mg/ml-distilled water) was treated with 1.7 mg of proteinase K (56 unit, Nacalai Tesque, Inc., Kyoto, Japan) at 37 °C for 30 min. The residual antifungal activity of the treated sample (50 µl) was tested by an agar-spot assay using A. brasiliensis NBRC9455. As positive and negative controls, the crude extract not treated with proteinase K and proteinase K solution not containing the crude extract were used, respectively, in the agar-spot assay.

Analysis of the antifungal compound by mass spectrometry
Electrospray ionization mass spectrometry (ESI-MS) analysis was performed to determine the molecular weight of the antifungal compound. The purified antifungal compound (50.0 µg/ml) was introduced into a mass spectrometer (Waters Q-TOF Premier, Micromass MS Technologies, Manchester, UK) at a flow rate of 10.0 μl/min with the capillary voltage of 2.5 V, collision energy of 5.0 V, and source temperature of 100 °C.

Isolation and identification of Aneurinibacillus sp. YR247
To establish an effective microorganism for the control of fungal contamination, antagonistic bacteria were isolated from the deep-sea sediment taken at Sagami Bay, Japan, at a depth of 1171 m. One of them, strain YR247, exhibited a distinct antifungal activity against A. brasiliensis NBRC9455. Its 16S rRNA sequence consists of 1509 nucleotides. The results of phylogenetic analysis based on 16S rRNA gene sequence data are shown in Fig. 1; strain YR-247 clearly falls within the genus Aneurinibacillus and belongs to Firmicutes. The nucleotide sequence of the 16S rRNA gene of strain YR-247 was most closely related to those of Aneurinibacillus migulanus B0270 T (X94195) and Aneurinibacillus aneurinilyticus Murayama T (X94194) with similarities of 99% for both. The next closest level of similarity was Aneurinibacillus danicus DB4 T (AB112725) with 97% identity. The 16S rRNA sequence of strain YR247 was submitted to NCBI GenBank (accession no. LC110197).

Antimicrobial activity of the crude extract
Although we tried the detection of antifungal activity using culture supernatant of strain YR247, the antifungal activity against A. brasiliensis NBRC9455 was not detected (data not shown). Therefore, we prepared the crude extract from the bacterial cell pellet and examined the antifungal activity. As the results, the antifungal activity of the crude extract against A. brasiliensis NBRC9455 was detected. As shown in Table 1, the crude extract exhibited a wide antimicrobial activity against fungus A. brasiliensis NBRC9455, Grampositive bacterium S. aureus NBRC12732 T and Gram-negative bacterium E. coli NBRC3301 T , but not yeast C. albicans NBRC1594. The inhibitory zones remained the same for several days (data not shown). Antimicrobial activities by the solvent against test strains were not detected by the agar spot assay.

Effect of temperature, pH, and proteinase K on antifungal activity
To examine the thermostability, the crude extract containing the antifungal compound was incubated at different temperatures for 6 h. The residual antifungal activity was tested by an agar-spot assay (Fig. 2a). Treatment at 4-70 °C did not decrease the antifungal activities (inhibition zone: 19.0-20.3 mM), whereas the activity was only slightly detected after being treated at 80 °C. The activities were not detected when the crude extracts were treated at 90 and 100 °C. As shown in Fig. 2b, the antifungal activity was not affected at pH 2.0-12.0 after incubation at 4 °C for 12 h (inhibition zone: 13.5-16.3 mM). Although antifungal activity was detected without the proteinase K treatment (inhibition zone: 13.5 mM), the antifungal activity was not observed after being treated with proteinase K at 37 °C for 30 min (Fig. 2c).

Purification and mass analysis of the antifungal compound
Because the crude extract obtained from the YR247 cells stably exerted antifungal activity, it was further purified using silica gel chromatography and preparative thin-layer chromatography. Approximately 2.3 mg of the antifungal compound was purified from the YR245 cell pellet (13.5 g). The R f value of the antifungal compounds was 0.62 using acetone:distilled water (7:1, v/v) on TLC silica gel plates (Fig. 3a). As shown in the mass spectrum at negative ion mode of the purified fraction (Fig. 3b), the molecular weight of the antifungal compound was estimated to be 1167.9 because the molecular ion peaks at m/z 1167.4 and 582.7 probably correspond to the charged ion [M−H] − and the doubly charged ion [M−2H] 2− , respectively (Gibson et al. 1993;Tilvi and Naik 2007). The calculation of molecular weight is indicated as follows: The molecular weight of the antifungal compound was estimated as 1167.9 using the average of both values (1168.4 and 1167.4).

Discussion
Aneurinibacillus sp. YR247 was isolated from the deepsea sediment inside the Calyptogena community at a depth of 1171 m in Sagami Bay of Japan. The strain exhibited antifungal activity against A. brasiliensis NBRC9455.  Using the crude extract prepared from YR247 cells, the antimicrobial activities were detected against A. brasiliensis NBRC9455, Gram-positive bacterium S. aureus NBRC12732 T , and Gram-negative bacterium E. coli NBRC3301 T , but not yeast C. albicans NBRC1594. The antifungal compound from strain YR247 was stable at 4-70 °C and pH 2.0-12.0. The antifungal compound was shown to have a rather stable structure as it retained activity after incubation at high temperatures and over a wide pH range as well as bacteriocin-like inhibitory substance from Bacillus cereus ATCC 14,579 and antifungal peptides from Bacillus sp. BH072 (Risøen et al. 2004;Zhao et al. 2013). After treatment with proteinase K, the antifungal activity was not detected, indicating that the antifungal compound is a peptidic compound (Fig. 2c). Among the related strains, it was already reported that Aneurinibacillus migulanus ATCC9999 T produces the cyclic peptide gramicidin S (Berditsch et al. 2007a). Bacillus brevis produces a mixture containing linear peptides, namely, gramicidin A (valine-gramicidin A and isoleucine-gramicidin A), gramicidin B (valine-gramicidin B and isoleucine-gramicidin B), and gramicidin C (valinegramicidin C and isoleucine-gramicidin C) (Tang et al. 1992). B. brevis also produces cyclic peptides, namely, tyrocidine A, tyrocidine B, and tyrocidine C. It was also reported that those peptides exhibit antimicrobial activities against Gram-positive bacteria including S. aureus and B. subtilis and Gram-negative bacteria including E. coli (Danders et al. 1982). Additionally, iturin A-like peptides from Bacillus sp. BH072 exhibited antifungal activities against A. brasiliensis (previously called (A) niger), Pythium, and Botrytis cinerea and had no inhibition on E. coli and S. aureus (Zhao et al. 2013). Fengycin-like peptide and iturinlike peptides from Bacillus amyloliquefaciens LBM 5006 exhibited antifungal activities against Aspergillus spp., Fusarium spp., and Bipolaris sorokiniana (Benitez et al. 2010). Bacillomycin-like peptides from (B) subtilis B-FS06 inhibited the growth and spore germination of Aspergillus flavus (Zhang et al. 2008). Compared with related studies, we indicated that the antifungal peptide from strain YR247 is responsible for the anti-Aspergillus activity.
In the case of strain YR247, Table 1 suggests that the antifungal peptidic compound exhibits the similar bactericidal activities. In addition to these bactericidal activities, gramicidin S from A. migulanus ATCC9999 T exhibits antifungal activity against C. albicans (Kondejewski et al. 1996). However, in the case of strain YR247, although antifungal activity against A. brasiliensis NBRC9455 was detected, antifungal activity against C. albicans NBRC1594 was not detected (Table 1). Thus, the results suggest that the peptidic compound from strain YR247 is distinct from gramicidin S. The antifungal activity of iturin and related lipopeptides depends on the composition of the length of the lipid chain (Maget-Dana and Peypoux 1994; Moyne et al. 2001). We indicated antifungal peptide with molecular weight of 1167.9. We suggested that two methylene groups (-CH 2 -) in the aliphatic chain of the antifungal peptide is more than that of gramicidin S (MW 1140.5). The difference in antimicrobial activity against C. albicans may depends on the composition of the length of the aliphatic chain.
Action mechanism of antibacterial activity by gramicidin S is assumed to be the accumulation of significant amounts of gramicidin S in a membrane, resulting in the destruction of the membrane integrity and a leakage of alkaline metal cations and protons (Mogi and Kita 2009). Although gramicidin S seems to be useful as a biocontrol agent against the agricultural pathogens, the hemolytic activity of gramicidin S restricts its use to topical applications. In this study, we purified gramicidin-like novel antifungal peptide from the deep-sea bacterium. The hemolytic activity of the antifungal peptide from strain YR247 should be examined and antibacterial and antifungal spectra needs to be determined. We expect elucidation of the action mechanism of the antifungal peptide from YR247 is useful for development of the biocontrol agent against the food and agricultural pathogens.