Microbes Environ. Vol. 18, No. 3, 126–132, 2003
http://wwwsoc.nii.ac.jp/jsme2/
Characterization of Sphingomonas Species Found as Predominant Members in the Culturable Bacterial Community of a Green Pigment-Containing Sclerotium Grain from Mt. Myoko (Japan) Volcanic Ash Soil HIROYUKI OHTA1*, MICHIE YAGI1, JUNKO SUZUKI1,3, NOBUHIDE FUJITAKE2 and MAKIKO WATANABE3 1
Department of Bioresource Science, College of Agriculture, Ibaraki University, Ami-machi, Ibaraki 300–0393, Japan 2 Department of Biological and Environmental Science, Faculty of Agriculture, Kobe University, Kobe 657–8501, Japan 3 Interdisciplinary Graduate School of Science and Engineering, Tokyo Institute of Technology, Yokohama 226– 8502, Japan (Received April 7, 2003—Accepted June 5, 2003) A small spherical black fungal sclerotium grain from podzolic soils, which was tentatively identified as the resting body of Cenococcum graniforme, was assumed as the source of green polynuclear quinone pigments in P type humic acid (K. Kumada and H.M. Hurst, Nature 214: 631–633, 1967). To examine the presence of bacteria inside sclerotium grains collected from an Andosol profile in Mt. Myoko, central Japan, the grains were repeatedly washed, ultrasonicated and then cultured on diluted nutrient broth. The sum of recovered bacteria as colonyforming units from the wash and ultrasonicate fractions was 1.46´106 (g fresh weight)-1: 88% of the count in the wash fractions (assumably resulting from grain surface and attached soil) and 12% in the ultrasonicate (inside grain). Thirty-one bacterial strains were isolated from the ultrasonicate fraction and their 16S rDNA partial sequences were determined. The predominant group was the Alphaproteobacteria (71%), chiefly the Sphingomonas group (52%). Representative isolates of the Sphingomonas group were examined for their ability to grow on naphthalenesulfonic acids as a model compound of polycyclic aromatic hydrocarbons (PAHs) and also on several phenolic acids. None of the isolates tested utilized the model PAH but many of them used p-hydroxy benzoic, vanillic, p-coumaric and ferulic acids for growth. Based on these results, the relationship between the predominance of Sphingomonas and the chemical character of the sclerotium grain was discussed. Key words: sclerotium, Sphingomonas, green pigment, P type humic acid
Various quinone pigments are found in the soil environment and known to be metabolites of higher plants, soil fungi and lichens3,4,6,25). Kumada et al.11–13) reported that P type humic acid commonly present in podzolic soils and alpine grassland soils had a characteristic absorption band resulting from the presence of green pigment. This pigment, precisely the green fraction of P type humic acid (Pg), was characterized as being composed of 4,9-dihydroxyperylene* Corresponding author; E-mail:
[email protected], Tel: +81– 298–88–8684, Fax: +81–298–88–8525
3,10-quinone derivatives22). Furthermore, it was suggested that the source of Pg was small spherical black fungal sclerotia and this fungal species was tentatively identified as Cenococcum graniforme (currently Cenococcum geophilum) from their morphological characteristics10). Sclerotia are known as resting structures of ectomycorrhizal fungi and appear as a response to unfavorable environmental conditions such as desiccation. In a recent study on the distribution of Pg-containing sclerotium grains in volcanic ash soils from Mt. Myoko, sclerotium grains were detected in the surface A and buried A horizons of nonallophanic soils
Sphingomonas spp. from a Sclerotium Grain
(maximum density, approximately 2.5 g kg-1) but not in allophanic soils28). Recently, electron microscopic examinations revealed that the Pg-containing sclerotium grain was hollow and the internal structure was characterized by the organization of hexagon units composing the transverse wall, and further the presence of fungal mycelium-like and bacterium-like structures inside the grain27,34). In this study, we aimed to isolate bacteria harbored in the Pg-containing sclerotium grain and further to characterize isolates phylogenetically and biochemically.
Materials and Methods Sampling of soil and sclerotium grains Sclerotium grains were obtained from Tsubame soil (Fulvic Andosol, WRB/FAO-Unesco) of Mt. Myoko, Niigata Prefecture, Japan (36°54¢00¢¢ N, 138°8¢25¢¢ E, altitude: 1,330 m) as described previously27). The pH values of the sampled soil were 4.9 in H2O and 4.3 in 1 M KCl27). Sclerotium grains were found as black, hard, smooth spherical bodies with a diameter of 0.5–1.5 mm (Fig. 1). The grains were carefully picked up from the soil and any soil on the surface removed using tweezers. Some of the grains collected were tested for the presence of Pg by extracting with 0.1 M NaOH as previously described27). The elemental composition of the sampled grains was described in our recent reports27,28).
Enumeration of bacteria and fungi The dilution plating method was used to enumerate viable bacteria and viable propagules of fungi in the sclerotium grains. One hundred-fold diluted nutrient broth (DNB) agar18) and rose bengal agar medium were used for culturing bacteria and fungi, respectively. The nutrient broth (NB, pH 7.0) was composed of 1% (w/v) meat extract (Kyokuto Seiyaku, Tokyo, Japan), 1% (w/v) polypeptone (Nihon Seiyaku, Tokyo, Japan) and 0.5% (w/v) NaCl. The medium was solidified by adding 1.5% (w/v) agar. The rose bengal
Fig. 1. Photomicrographs of a sclerotia grain (indicated by an arrow) found in the soil (A) and washed sclerotia grains (B). Bar, 1 mm.
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agar medium (pH 6.8) contains (g per liter): KH2PO4 1.0, MgSO4·7H2O 0.5, peptone 5.0, glucose 10.0, rose bengal 0.033, and agar 20. To remove surface-attached soil, a sclerotium grain was washed in a microtube containing 1 ml of sterile water with 1 min-vortex mixing then the washing solution was removed from the tube with a sterile pipette. After ten such washes, the grain was crushed with a sterile glass rod in 1 ml of sterile water and then ultrasonicated for 2 min at 100W. Next, 0.1 ml of the suspension or 10-fold diluted suspension was pipetted onto the agar medium and spread with a sterile glass rod. The plates were incubated for 3 or 4 weeks at 27°C before the counting of colony-forming units (CFU). Duplicate plates were prepared for each grain sample.
Isolation and cultural characteristics of bacteria For the isolation of bacteria, colonies on agar plates were chosen randomly for subculture on DNB agar plates and bacterial cells were reisolated from colonies on the plates after checking culture purity. Isolates were examined for cell morphology and growth on full-strength nutrient broth (NB) and 10-fold diluted NB as previously described18,19). The effect of pH on the growth was determined using 10fold diluted NB liquid cultures (2 ml) with a pH value ranging from 3.1 to 7.7. The cultures were stood at 27°C for 4 days and then aliquots (150 ml) from the cultures were measured for optical density at 490 nm (OD490) in a microwell plate on a PerkinElmer-Wallac 1420 ARVOsx multi-label counter.
Phylogenetic characterization of isolates Genomic DNA was extracted from bacteria grown on DNB agar plates by the method of Wang and Wang26) for the amplification of the 16S rDNA via PCR with the primers fD1 (Escherichia coli positions 8–27) and rD1 (E. coli positions 1542–1525)29). The PCR conditions were as follows: a hot start at 96°C for 5 min then 25 cycles consisting of 30 s at 96°C, 1 min at 60°C and 1 min at 72°C. All amplifications were done in a DNA thermal cycler (model 480, Perkin Elmer). The amplified DNA was analyzed by electrophoresis on 1% agarose gels with TBE buffer15) and the gels were stained with ethidium bromide. The bands were visualized by UV excitation to check the size of the amplification product. The PCR products were purified by polyethylene glycol precipitation14). Nucleotide sequences were determined using the Taq Dye Deoxy Terminator Cycle Sequencing kit (Perkin Elmer) and read on an Applied Biosystems 373A DNA sequencer. The primer f3L (E. coli positions 1094–1112)8) was used in sequencing reactions to
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obtain partial DNA sequences. All sequences determined were compared to similar DNA sequences retrieved from the DDBJ/EMBL/GenBank databases by using the BLAST program20). The DNA sequences were aligned using the CLUSTAL W program24) and a phylogenetic tree was created by the neighbor-joining method21).
Biochemical characterization of isolates The assimilation of organic compounds including carbohydrates and organic acids was tested using API 20 NE (bioMérieux). The tests were run as instructed by the manual and incubated at 27°C for 4 days. To examine the degradation profile of aromatic compounds, bacterial isolates were grown at 27°C in DNB liquid cultures supplemented with one of the following compounds (2 mM): phenol, benzoic acid, p-hydroxy benzoic acid, protocatechuic acid [3,4-dihydroxybenzoic acid], vanillic acid [4-hydroxy-3methoxybenzoic acid], p-coumaric acid [3-(4-hydroxyphenyl) -2-propenoic acid], caffeic acid [3-(3,4-dihydroxyphenyl) -2-propenoic acid], ferulic acid [3-(4-hydroxy-3-methoxyphenyl)-2-propenoic acid], p-toluenesulfonic acid [4-methylbenzenesulfonic acid], 1-naphtalenesulufonic acid and 2,6naphthalenedisulfonic acid. These acids were prepared as sodium salts. The cultures (3 ml) were stood for 1 week. Aliquots (150 ml) from the cultures were measured for OD490 in a microwell plate on the multi-label counter. Degradation of a compound was judged by both an increase in OD490 in comparison with a control DNB culture and a decrease in UV absorption.
Nucleotide sequence accession numbers The 16S rDNA sequence data for isolates in this study have been deposited under DDBJ accession numbers AB107136 to AB107198.
Results and Discussion Isolation of bacteria from a sclerotium grain Effects of repeated washes and the ultrasonication on plate counts of bacteria from the sclerotium grain were examined. The results of three independent experiments are shown in Fig. 2. The first wash fraction of sample A (fresh weight, not determined) yielded a plate count of 3.05´103 CFU. The relative plate counts for the third and tenth wash fractions were only 3.6% and 0.7%, respectively, of the former. Following the wash treatment, the ultrasonication gave 7.9% of the plate count of the first wash fraction, suggesting a large release of bacteria from the inside of the sclerotium. This was also the case for the second sample
Fig. 2. Plate counts of repeated wash solutions and ultrasonicates of sclerotia grains sampled from Mt. Myoko volcanic ash soil. Sclerotium samples (fresh weight): , A (not determined); , B (4 mg); , C (9 mg).
(sample B; fresh weight, 4 mg): the relative plate counts of the tenth wash fraction and the ultrasonicate fraction were 0.5% and 24%, respectively, of the total recovered CFU from the wash and ultrasonicate fractions. According to the same procedure, sample C (fresh weight, 9 mg) was fractionated (Fig. 2) and then bacteria were isolated from the ultrasonicate and the first wash fractions. The sum of recovered bacteria as CFU from sample C was 1.46´106 (g fresh weight)-1: 88% were found in the wash fractions and 12% in the ultrasonicate. Counts of fungal propagules were 1.47´105 (g fresh weight)-1 (86%) in the wash fractions and 2.40´104 (g fresh weight)-1 (14%) in the ultrasonicate.
Phylogenetic analysis of sclerotium bacteria Thirty-one and 37 bacterial strains were isolated from DNB plates of the ultrasonicate and the first wash fraction, respectively, from sample C. Partial 16S rDNA sequences of 63 successfully subcultured strains, 29 from the ultrasonicate and 34 from the first wash fraction, were determined. The DNA sequences determined were compared to similar sequences retrieved from the DDBJ/EMBL/GenBank databases. Table 1 represents the phylum (or class) level phylogenetic diversities of bacterial isolates. The first wash fraction contained Proteobacteria (59%), Actinobacteria (22%), and Firmicutes (8%) as the predominant groups. This composition was analogous to the bacterial profiles found in most environmental soils1,16). In contrast, the ultrasonicate fraction was dominated by the Proteobacteria (84%), chiefly the Alphaproteobacteria (71%). Figure 3 illustrates a phylogenetic tree based on the partial 16S rDNA
Sphingomonas spp. from a Sclerotium Grain Table 1.
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Phylum (or Class) level phylogenetic diversity identified in 16S rDNA of isolates from the first wash solution (surface fraction) and ultrasonicate (inside fraction) of a sclerotium grain in Mt. Myoko Tsubame soil. No. of isolates (%) from
Phylum/Class First wash fraction Ultrasonicate fraction Proteobacteria Alphaproteobacteria Betaproteobacteria Gammaproteobacteria Firmicutes Actinobacteria Bacteroidetes Not identified
22 (59) 9 (24) 12 (32) 1 (3) 3 (8) 8 (22) 1 (3) 3 (8)
26 (84) 22 (71) 4 (13) 0 3 (10) 0 0 2 (6)
Total
37a (100)
31b (100)
a
Isolates from the first wash fraction were labeled W and the DDBJ accession numbers for their 16S rDNA sequences are AB107165– AB107198. b Isolates from the ultrasonicate fraction were labeled K and the DDBJ accession numbers for their 16S rDNA sequences are AB107136– AB107164.
sequences of the 29 isolates from the ultrasonicate and those of related strains retrieved from the databases. Among the 22 strains belonging to the Alphaproteobacteria, 16 strains (52% of all isolates from the ultrasonicate) were placed in 5 different clusters of the sphingomonads: clusters A (number of strains, 2), B (3), C (2), D (1), and E (8). Recently, on the basis of a phylogenetic analysis of 16S rDNA sequences and polyamine profiles, it was proposed that the genus Sphingomonas be classified into four genera, Sphingomonas sensu stricto, Novosphingobium, Sphingobium, and Sphingopyxis23). Based on this proposal, the cluster A strains corresponded to Novosphingobium, the cluster B strains to Sphingopyxis, the cluster C strains to Sphingomonas sensu stricto, and the cluster E strains to Sphingobium. Strain K-2059 was closely related to Sphingomonas natatoria32) (cluster D) whose basonym is Blastomonas natatoria9). Hiraishi et al.9) and Takeuchi et al.23) proposed that Blastomonas and Rhizomonas could be distinguished from all Sphingomonas species at the generic level by the combined use of chemotaxonomic and phenotypic markers. With respect to the proposal of Takeuchi et al.23), Yabuuchi et al.31) reported that there is no phenotypic and phylogenetic evidence to support the proposal to split the species into four genera and concluded that the genus Sphingomonas should remain undivided at this time. Contrary to this, very recently, Busse et al.2) argued that the presence of homospermidine should be considered as a distinguishing charac-
ter within the family Sphingomonadaceae and strongly supported the proposal to retain the genus Sphingomonas only for the members of cluster I according to Takeuchi et al.23)
Biochemical characteristics of Sphingomonas isolates from sclerotium A number of bacteria belonging to the Sphingomonas group are known to have unique abilities to degrade polycyclic aromatic hydrocarbons (PAHs). For example, Sphingomonas aromaticivorans strain F199 can grow on biphenyl, naphthalene and dibenzothiophene5,7). Sphingomonas yanoikuyae strain B1 can grow on biphenyl, naphthalene, phenanthrene and anthracene35). Sphingomonas paucimobilis strain EPA505 utilizes fluoranthene, naphthalene and phenanthrene as the sole carbon and energy sources17). In relation to this, it is of note that the sclerotium grain contained polynuclear quinones, 4,9-dihydroxyperylene-3,10-quinone derivatives22). Therefore, the predominance of Sphingomonas in the sclerotium grain might be explained by the presence of specific nutrients for the organisms. Because polynuclear quinones are not commercially available, a bicyclic aromatic compound, naphthalenesulfonic acid, was chosen as a model compound and representative strains (K2036, K-2055, K-2062, K-2059 and K-2056) from each of the clusters were examined for the ability to degrade it. However, none of the strains tested degraded 1-naphtalenesulufonic acid or 2,6-naphthalenedisulfonic acid. The ability to degrade aromatic compounds was further tested with benzoic acid, p-toluenesulfonic acid, phenol and some phenolic acids (p-hydroxy benzoic acid, vanillic acid, caffeic acid, p-coumaric acid and ferulic acid). Except for strain K2055 (cluster B), all of the other strains utilized p-hydroxy benzoic acid, vanillic acid, p-coumaric acid and ferulic acid (Table 2). In addition to these compounds, strain K-2062 (cluster C) used benzoic acid and caffeic acid, and strain K2036 (cluster A) caffeic acid. However, none of the strains utilized phenol and p-toluenesulfonic acid. The API 20 NE test showed that the Sphingomonas isolates examined did not assimilate glucose and malate. Utilization of glucose was found in not only the reported PAH-degrading S. yanoikuyae and S. paucimobilis33) but also a number of strains of Sphingomonas group33). Therefore, the negative response of the isolates to glucose suggests a highly specialized feature for the utilization of phenolic acid compounds and might explain the predominance of the organisms in the sclerotium grain when phenolic compounds would represent intermediates of polynuclear quinone pigment degradation. Previously, the chemical composition of the same sclerotium grains used in this study was determined with an
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Fig. 3. Unrooted tree showing the phylogenetic relationships of the isolates from the ultrasonicate of a sclerotium grain and their related species in the Alphaproteobacteria, the Betaproteobacteria and Paenibacillus taejonensis and Bacillus cereus. The tree, constructed using the neighbor-joining method, was based on aligned sequences of the 16S rDNA (positions 1141–1475 of the nucleotide sequence of Escherichia coli). Bootstrap values, expressed as a percentage of 1000 replications, are given at branching points. Scale bar, 5 nucleotide substitutions per 100 nucleotides.
energy dispersive X-ray spectrometer and a NC analyzer28). The values of total C%, N%, and C/N were 31.2–50.4%, 1.0–1.8%, and 22.2–35.1, respectively. The weight percentage (excluding C and N) of Al2O3 and SiO2 was 48.3–81.4 and 0.0–9.8%, respectively. Interestingly, the mean weight of the sclerotium grains tended to increase in soils with an exchangeable aluminum (Al3+) content higher than 2 cmolc kg-1, suggesting that the development of the sclerotium
grains is influenced by the aluminum status in soils. Hence, bacteria inside the sclerotium grain might be exposed to different levels of aluminum and the Al level may represent a selective stress for the growth or survival of bacteria in the grain. As the pH decreases, aluminum becomes more soluble in the soil solution and toxic to plants and microorganisms. Therefore, prior to testing Al tolerance, we examined the effect of acid stress on the growth of our Sphingomonas
Sphingomonas spp. from a Sclerotium Grain Table 2.
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Growth and biochemical characteristics of Sphingomonas strains isolated from the ultrasonicate (inside fraction) of a sclerotium grain in Mt. Myoko Tsubame soila. Sphingomonas strains (cluster)b Characteristics
Growth on NB NB/10 Assimilation of c: Benzoic acid p-Hydroxy benzoic acid Protocatechuic acid Vanillic acid p-Coumaric acid Caffeic acid Ferulic acid Activity of: Arginine dihydrolase Urease b-Galactosidase Aesculin hydrolysis Gelatin hydrolysis
2036 (A)
2055 (B)
2062 (C)
2059 (D)
2056 (E)
+
+ +
+ +
+ +
+ +
+ + + + + +
-
+ + + + + + +
+ + + + +
+ + + + +
+ + + + +
+ + -
+ + -
+ + -
+ -
a
Symbol: +, positive reaction; -, negative reaction. Clusters A–E, see Fig. 3. c None of the strains tested assimilated D-glucose, malic acid, p-toluenesulfonic acid, naphthalenesulfonic acid or phenol. b
unlikely that our Sphingomonas isolates have remarkable tolerance to aluminum toxicity. Many strains of Sphingomonas have been isolated from waters, soils, plants and clinical specimens30), but an exact picture of the ecology of Sphingomonas has not yet been obtained. In this study, we focused on the microbial community of chemically well-characterized microenvironments such as sclerotium grains and found a predominance of the Sphingomonas group. Further studies are now in progress on the distribution of Sphingomonas and microbial communities in fungal sclerotia from different soil layers and different soils.
Acknowledgements Fig. 4. Effect of medium pH on the growth of representative strains of Sphingomonas spp. isolated from a sclerotium grain. Strain (cluster): , M2036 (A); , M2055 (B); , M2062 (C); , M2059 (D); , M2056 (E).
This work was supported in part by Grants-in-Aid (No., 13680102) for Scientific Research from the Japan Society of the Promotion of Science.
isolates in the absence of aluminum. As shown in Fig. 4, none of the representative strains showed acid tolerance and grew at the pH values below 4–5. From this result, it is
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