Isolation, characterization and identification of oligotrophic strain KP1-77T is

Abstract.[Objective] We characterized an oligotrophic growth bacterium KP1-77T, which was isolated from 22-year-old volcanic deposits of Miyake-jima island (Japan) in this study. [Methods] Oligotrophic growth for strain KP1-77T was tested using nutritionally deficient medium. N2-fixing capability was examined using NFb medium. Phenotypic tests was mainly done with API 20NE kit system (bioMérieux). Phylogenetic position was determined by constructing the system tree. Cellular fatty acids were extracted with 5% HCl-methanol and analyzed with GC-14B gas chromatography. DNA G+C contents were measured with DNA GC kit and reversed-phase HPLC. DNA-DNA hybridization was carried out with photobiotin-labelled probes in microplates wells. [Results] Oligotrophic growth of strain KP1-77T was proved by its least multiplication number of 4.2×105 cfu/ml cultured in series of diluted NB medium and distilled water. N2-fixing capability was determined by its obvious growth on NFb medium. Strain KP1-77T was Gram-negative and slight curved rod (0.3-0.5×3-8μm). The optimum growth occurred at the temperature ranging from 27°C to 37°C. Strain KP1-77T can assimilate glucose, L-arabinose, D-mannose, D-mannitol, N-acetyl-D-glucosamine, maltose, caprate, adipate, dL-malate and phenylacetate, not gluconate and citrate. The phylogenetic analysis based on 16S rDNA gene sequences indicated that it was clustered in the genus Herbaspirillum, and the sequence similarity of 98% to Herbaspirillum seropedicae LMG 6513T has been detected. The major cellular fatty acids are C16:0(29.3%), C16:1w7c/C15:0 2-OH iso (36.5%), C18:1 iso/trans (22.7%). The DNA G+C content of strain KP1-77T was 61mol%. DNA-DNA relatedness between strain KP1-77T and H.seropedicae LMG6513T, H.putei NBRC102406T and H.rubrisubalbicans NBRC102523T were 18.7%, 20.7% and 17.5% respectively. [Conclusions] As for the above results, strain KP1-77T seemed to be a novel species in the genus Herbaspicrillum with its accession number of AB366211 and the name of Herbaspirillum scorialis sp. nov. was proposed.

Key words: volcanic soil;bacterium isolation; oligotrophic; novel species

Volcanic deposits have been regarded as an ideal model ecosystem to investigate its development[1-3]. Miyake-jima island (55.14 km2, 814.5 m in height), is an active volcano island, situated in the western rim of the Pacific Ocean (34° 05’ N, 139° 31’E), about 180 km south of Tokyo. For this island, the eruptions of 1874-y, 1940-y, 1962-y, 1983-y, and 2000-y were recorded in the recent history. Land covered with lava from the series of eruptions was suited to a chronosequence study of the bacteria community structures. Despite ecological research on vegetation on this island has been done much, very little is known about microbial species and functional diversity in the volcanic deposits. In addition, the information about the influence of the microorganisms on sustainable ecosystems is also very limited.

In the present research work, we chose the 23-year-old volcanic deposit at Nippana site (34°02’50.7’’N, 139°30’02.1’’E) on the island of Miyake as our sample. The bacterial community in those volcanic deposits was analyzed by culture-based method and the representative isolate was tested on its physiological and biochemical characteristics.

1.Materials And Methods

1.1.Samples description and bacteria isolation

The volcanic deposits were sampled (area of 15×15cm; depth of 0 to 7 cm) from the unvegetated site KP1 (34°02′50.77″N, 139°30′02.1″E), at the distance of 20 m from the sea shore in the south of Mike-kejima island. Collected samples were stored in plastic bags and kept at 4 ℃ until the bacteriological analysis.

Total organic carbon was analyzed using a Shimadu TOC-V total organic carbon analyzer according to the manufacture’s instructions. Total carbon and nitrogen were measured on a Yanaco CHN Corder type MT-6 (Yanaco Analytical Instruments Corp., Kyoto, Japan). Slurries (1:2.5 mass ratios of samples and deionized water) were used to determine pH. The sample water content was estimated by drying material at 105oC overnight. The enumeration and isolation of culturable bacteria were carried out with 100-fold diluted nutrient broth (DNB) agar medium [5].

1.2.Phenotypic tests of strain KP1-77T

Cell morphology and Gram stain for the pure cultures of isolates were examined as previously described [5]. Growth temperature range was tested by incubating the isolates in 10-fold diluted nutrient broth (10-1 NB) liquid medium at 4℃, 27 ℃, 37℃ and 40℃ for a two-week period. The pH range (pH 4~10, using increments of 2 pH unit) for growth was determined by assessing changes in OD550 over the incubation time (up to 7days) in 10-1 NB medium [6]. The final pH was adjusted using NaOH and HCl solutions. Other phenotypic characteristics of strain KP1-77T were examined by the methods of Ohta & Hattori[5] and API 20NE kit system (bioMérieux) according to the recommendation of the manufacture. N2-fixing activity was examined by incubating strain KP1-77T at 30 ℃ on semi-solid NFb medium containing the following ingredients (gL-1): KH2PO4, 1.2; KHPO4.0.8; potassium malate, 5.0; MgS04.7H2O, 0.2; NaCl, 0.2; CaCI2・2 H2O, 0.02; FeSO4.7 H2O, 0.002; together with trace element solution, 2mlL-l; and biotin, 10 ugL-1. The trace element solution contained (gL-1): Na2MoO4.2H2O, 1.0; MnSO4.H2O, 1.75; H3BO3,1.4; CuSO4.H2O, 0.04; and ZnSO.7H2O, 0.12.

1.3.Oligotrophic growth test

Strain KP1-77T was cultured on the 100-fold diluted nutrient broth (NB) liquid medium at 30°C for 5 days. The obtained culture was sub-cultured on the NB medium, 100-fold diluted NB medium, 1000-fold diluted NB medium, 10000-diluted NB medium and distilled water three times respectively at 30°C for 5 days. Then those series of cultures obtained from the above were incubated on 100-fold diluted nutrient broth (NB) argar solid medium respectively at 30°C for 7 days and the colonies appeared on the plates were counted. Three parallel tests were done for all the previous sub-cultue and colonies counts.

1.4.Phylogenetic analysis of KP1-77T

Genomic DNA of the bacterial cells cultured on DNB agar plates was extracted according with the protocol of Wang and Wang [7]. Polymerase chain reaction (PCR) amplification of 16S rRNA of each isolate was carried out using the following primers 10F (Escherichia coli positions 10-27) and 1541R (E. coli positions 1541-1521) [4]. The PCR conditions and the purification of PCR products were essentially the same as described previously [4]. Amplified nucleotide sequences were determined by ABI PRISMTM Big Dye Terminator Cycle Sequencing kit (Applied Biosystems, Foster, USA) and read on an Applied Biosystems 3100 DNA sequencer. The gene sequences were compared with similar DNA sequences retrieved from the DDBJ/EMBL/GenBank databases using the BLAST program[9]. For phylogenetic analysis of the sequence datasets, the CLUSTAL W program [10] was utilized and a tree was constructed by the neighbor-joining method [11]

1.5.Fatty acids components

Cellular fatty acids were extracted with 5% HCl-methanol, as described by Ikemoto et al., [12]. Fatty acids profiles were analyzed by a model GC-14B gas chromatography (Shimadzu Corp., Kyoto, Japan) equipped with a capillary column ULBON HR-SS-10 (0.23mm×50m:Shimadzu Corp., Kyoto, Japan) and a hydrogen flame ionization detector.

1.6.DNA G+C contents and DNA-DNA hybridization

To determine the DNA G+C contents, genomic DNA was prepared according to the method of Saitou & Miura [13] and digested with P1 nuclease using the DNA GC kit (Yamasa Shoyu). The G+C contents were determined by reversed-phase HPLC as described by Tamaoka & Komagata [14].

DNA-DNA hybridization was carried out with photobiotin-labelled probes in microplates wells, as described by Ezaki et al. [15]. Alkaline phosphatase-streptavidin conjugate (Vector) was used with CDP-Star (Tropix) as substrate and Wallac 1420 ARVOsx multilabel counter was used for the determination of chemiluminescence as described by Ushiba et al. [16]

1.7.Nucleotide sequence accession numbers

Accession numbers of the partial 16S rRNA gene sequences for isolates were obtained by depositing partial sequences with GenBank of National Center for Biotechnology Information (www.ncbi.nih.gov). The accession numbers (in parenthesis) for the isolates were shown in the phylogenetic trees.

2.Results

2.1.Chemical analysis of volcanic deposit samples of KP1

The total organic carbon (TOC) content of KP1 was 0.004%. Total carbon and nitrogen were 0.03% and

2.2.Phenotypic characteristics of Herbaspirillum-related strain KP1-77T

Strain KP1-77T was a Gram-negative slight curved rod (0.3-0.5×3-8μm). Growth was not found at 4°C and 40°C. The optimum growth occurred at the temperature ranging from 27 to 37°C. The nitrogen-fixing ability of strain KP1-77T was proved by its growing in the semi-solid NFb medium. Strain KP1-77T can assimilate many variable carbohydrates and organic compounds including glucose, L-arabinose, D-mannose, D-mannitol, N-acetyl-D-glucosamine, maltose, caprate, adipate, dL-malate and phenylacetate, not gluconate and citrate. Other physiological characteristics of strain KP1-77T and several related Herbaspirillum species are given in Table.1.

Table 1. Biochemical test for strain KP1-77T and reference Herbaspirillum species with API 20NE (bioMérieux) system. Strains: 1, KP1-77T; 2, H. seropedicae LMG 6513T; 3, H. hiltneri LMG 23131T; 4, H. putei 102406T; 5, H. huttiense 102521T; 6, H. rubrissuba 102523T; 7, H. chlorophenolicum 102525T

2.3.Oligotrophic growth

2.4.Phylogenetic position of strain KP1-77T in genus of Herbaspirillum

Phylogenetic analysis based on 16S rDNA sequences shows that strain KP1-77T is a member of the genus Herbaspirillum, and the most closely related species is H. seropedicae with the 16S rDNA sequence identity of 98% (Fig. 2).

2.5.Tests of fatty acids profiles, DNA-DNA hybridization and G+C contents

Reference strains of H. seropedicae LMG 6513T and H. hiltneri LMG 23131T were obtained from BCCM/LMG Bacteria Collection, Laboratorium voor Microbiologie, Gent, Belgium. H. autotrophicum NBRC 13527T, H. lusitanum LMG 21710T, H. putei NBRC 102406T, H. huttiense NBRC 102521T, H. frisingense NBRC 102522T, H. rubrisubalbicans NBRC102523T, and H. chlorophenolicum NBRC 102525T were obtained from National Institute of Technology and Evaluation Biological Resource Center, Gent, Belgium, Japan.

The major cellular fatty acids of C 16:0(29.3 %), C 16:1 w7c/C 15:0 2-OH iso (36.5%) and C 18:1 iso/trans(22.7%) have been detected for strain KP1-77T. The domain fatty acids were similar to other Herbaspirillum species with only differences in proportions. The fatty acids profiles were summarized in Table.2

Table 2. Fatty acids profiles of strain KP1-77T and Herbaspirillum reference species. 1, strain KP1-77T; 2, H. seropedicae LMG6513T; 3, H. hiltneri LMG23131T; 4, H. putei NBRC102406T; 5, H. huttiense NBRC102521T; 6, H.rubrisubalbicans NBRC102523T; 7, H. chlorophenolicum NBRC102525T

The DNA G+C contents was 61 mol%, fell within the range of 57.9%-65% of this genus. The values for hybridization of isolate KP1-77T with H. seropedicae LMG 6513T and with H.putei NBRC102406T and with H.rubrisubalbicans NBRC102523T were 18.7%, 20.7% and 17.5 %respectively. Such relatedness was low enough for strain KP1-77T to be classed into a novel Herbaspirillum species[17]. NifH gene was detected by PCR approach and the further sequence analysis based on nifH genes showed a similarity of 97% to its closest species of Herbaspirillum seropedicae.

3.Discussion

In our study, strain KP1-77T, as a represent of the predominant bacteria colonizing at Miyake-jima island, has been proved for its oligotrophic growth and N2-fixing ability. It seems to be one of the possible explainations on how those microorganisms could survive in such new volcanic ashes with extremely deficient nutrient. In the previous study on Miyake-jima island, Limnobacter litoralis KP1-19 T was also isolated from the same KP1 site and its oligoutrophic growth and thiosulfate oxidizing ability have been reported by Lu et al. in 2008[18] . This result was very concordant with the oligotrophic growtht of strain KP1-77T. Concerning the ecosystem development of KP1 site, the appearance of the plants and variable microorganism in this area were probable due to those oligotrophic and chemolithoautorophic bacteria which colonized in those new formed volcanic ashes with very low nutrients. Ademan[19] reported that reduced sulfur gases such as dimethylsulfide, carbonyl sulfide, and carbon disulphide are emitted from coastal sea water into the atmosphere. Because our site KP1 is very near the sea shore, the first colonized bacteria may be assimilate those sulfur compounds and nitrogen in the air as their energy source or nutrients and survived in those low nutrient volcanic ashes which was formed just after the eruption of the Miyake-jima island.

4.Acknowledgements

This work was supported in part by a Grant-in-Aid (No. 17310018) for Scientific Research from the Japan Society for the Promotion of Science. This study was performed in Ohta Hiroyuki’s lab. of Environmental Microbiology, University of Ibaraki, Japan.

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