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Isolation and Characterization of An Alkaliphilic and Halotolerant Nesterenkonia.sp from An Extreme Soda Saline-alkali Soil in the Northeastern of China | Shi 1 | Molecular Soil Biology

Isolation and Characterization of An Alkaliphilic and Halotolerant Nesterenkonia.sp from An Extreme Soda Saline-alkali Soil in the Northeastern of China  

Wei Shi1 , Takano Tetsuo2 , Shenkui Liu1
1. Key Laboratory of Saline-alkali Vegetation Ecology Restoration in Oil Field (SAVER), Ministry of Education,; Alkali Soil Natural Environmental Science Center (ASNESC), Northeast Forestry University, Harbin Hexing Road, 150040;
2. Asian Natural Environmental Science Center(ANESC), University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo, 188-0002, Japan
Author    Correspondence author
Molecular Soil Biology, 2012, Vol. 3, No. 2   doi: 10.5376/msb.2011.03.0002
Received: 29 Jun., 2011    Accepted: 01 Aug., 2011    Published: 09 Aug., 2011
© 2012 BioPublisher Publishing Platform
This is an open access article published under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Preferred citation for this article:

Shi et al., 2011, solation and characterization of an alkaliphilic and halotolerant Nesterenkonia.sp from an extreme soda saline-alkali soil in the northeastern of China, Molecular Soil Biology, doi:10.5376/msb.2011.03.0002

Abstract

A strain of alkaliphilic and halotolerant, rod-shaped bacterium, designated strain SA-3, was solated from an Extreme soda saline-alkali soil in Heilongjiang, northeastern China. Strain SA-3 was Gram-positive, catalase-positive, and oxidase-negative and formed Orange colonies, and starch is not hydrolyzed, optimal growth conditions were pH 9.0 and 0.5 mol/L NaCl. Strain SA-3 has strong resistance to Na2CO3, hight pH value, and NaCl. 16S rRNA a gene sequence alignment analysis show that the strain formed a separated line within a clade containing the genus Nesterenkonia in the phylum Actinobacteria and was related to the species Nesterenkonia lutea YIM 70081T (sequence similarity 99.2%), Nesterenkonia sandarakina YIM 70009T (99.2%), Nesterenkonia jeotgali JG-241T (99.1%), Nesterenkonia halotolerans YIM 70084T (99.0%), Nesterenkonia aethiopica DSM 17733T (97.5%). This similarity, suggest that this salt-tolerant alkaliphilic bacterium belonged to genus Nesterenkonia. This strain is a great potential microbial resources for development.

Keywords
Nesterenkonia; 16S rDNA sequence

Nesterenkonia as a genus was put forward by Stackebrandt (Stackebrandt et al., 1995). Which be classified as which be classified as one genus of common bacterial form and hight G+C content Actinobacteria, Currently, the genus contai eleven species: Nesterenkonia halobia, Nesterenkonia lacusekhoensis, Nesterenkonia halotolerans, Nesterenkonia xinjiangensis, Nesterenkonia lutea, Nesterenkonia sandarakina, Nesterenkonia aethiopica, Nesterenkonia jeotgali, Nesterenkonia halophila and Nesterenkonia flava, Nesterenkonia alba (Luo et al., 2009). Most of this genus, were isolated from hypersaline or alkaline environments, maily are the actinomycete group with the ability of alkali resistance properties.

As enzymes and antibiotics resources, Alkaliphilic actinomycetesget more attention of scholars dedicated to research in this area, and find several new agents and antibiotics. In order to develop the more effective use of such extreme environmental actinomycetes, we surveyed the halophilic bacteria in the regions of no perennial plant covering and pH 10.5 of extreme soda saline-alkali soil lands in Heilongjiang, northeastern China. A sum of 52 aerobic halophile strains was isolated from there, including three actinomycetes, one was identified from in the growth of actinomycetes under the pH 11.0 conditions. According to morphological, phenotypic characteristics and 16S rDNA sequence analysis, we identified taxonomy and phylogenetic position of this strain.

1 Results and Analysis
1.1 Phylogenetic analysis
Strain SA-3 was Gram-positive, catalase-positive, and oxidase-negative, starch is not hydrolyzed, mean size 0.56 μm×0.73 μm (Figure 1A). Colonies were circular, convex, moist, opaque, 7.5 mm wide, smooth and Orange on medium I (Figure 1B).

 
Figure 1 Scanning electron microscope (×10 000) and colony photos of strain SA-3

1.2 Resistance characteristics of the analysis

E.coli JM109 as a control, the strain SA-3 was used in the analysis of resilience analysis showed (Figure 2): In the tough environment of Na2CO3, strain SA-3 growth occurred in media containing 0~250 mmol/L Na2CO3 and optimal growth were observed in media containing 30~100 mmol/L Na2CO3. On the condition of Na2CO3 not lower than 30 m we can't find E.coli JM109 grows; In the tough environment of NaCl, strain SA-3 growth occurred in media containing 0~2.0 mol/L NaCl and optimal growth was observed in media containing 0~0.5 mol/L. E.coli JM109 was grew in the media containing 0.0~0.5 mol/L NaCl, which is not grown in the media containing up to 0.5~2.0 mol/L NaCl; In the tough environment of pH value, strain SA-3 grew at pH 7.0~11.0, that showed optimal growth was pH 8.0~9.0, E.coli JM109 grew at pH 7~8, severely inhibited the growth of E.coli JM109 grew at pH 9~12. In short, in the tough environment of Na2CO3, pH value, NaCl, the strain SA-3 was better than E.coli JM109 on the salt and alkali resistances.

 
Figure 2 Resilience analysis of strain SA-3 and E.coli JM109

1.3 Phylogenetic analysis

Phylogenetic alignment analysis of the partial 16S rRNA gene sequence (GenBank databases under Accession Number JF937438) showed that the isolated SA-3 in a distinct lineage with Nesterenkonia lutea YIM 70081T within the genus Nesterenkonia (Figure 3). These partial 16S rRNA gene sequences analysis suggested that strain SA-3 was most closely related to Nesterenkonia lutea YIM 70081T (sequence consistency: 99.2%), Nesterenkonia sandarakina YIM 70009T (99.2%), Nesterenkonia jeotgali JG-241T (99.1%), Nesterenkonia halotolerans YIM 70084T (99.0%), Nesterenkonia aethiopica DSM 17733T (97.5%), the highest similarity, higher than 97%. The results show that the strain SA-3 is belonged to genus Nesterenkonia.

 
Figure 3 16S rRNA sequence phylogenetic tree

Summary, the strain SA-3 was isolated from Extreme soda saline-alkali soil. It was Gram-positive, catalase-positive, and oxidase-negative and formed Orange colonies, and starch is not hydrolyzed, which is a member of the genus Nesterenkonia. In E.coli JM109 as a control, in the tough environment of Na2CO3, pH value, NaCl, the strain SA-3 was better than E.coli JM109 on the salt and alkali resistances. The strain SA-3 is salt-tolerant alkaliphilic actinomycetes. The salt-tolerant alkaliphilic actinomycetes is a particular taxa of actinomycete grow in extreme environment, which can produce alkaline proteases, amylases, cellulases and lipases that having unique properties under extreme conditions. These biocatalysts Widely used in industrial production. Wherefore the strain SA-3 have a great potentiality to develop.

2 Materials and Methods
2.1 Micro-organisms and culture conditions
In the couse of the survey of cultured bacteria of alkaline environments in heilongjiang, northeastern China, strain H1 was isolated from a soil sample collected from the Extreme soda saline-alkali soil in Songnei Plain. The strain was isolated by the standard dilution-plating technique based on medium â…  ( KCl, 2.0 g; MgSO4×7H2O, 1.0 g; NaCl, 30 g; Na3-citrate, 3.0 g; yeast extract, 10.0 g; (1 mL/L of g/L) MnCl2×4H2O, 0.36 g and FeSO4, 50 g; agar, 15 g; distilled water, 1 000 mL, Media adjusted to the pH values 9.0) at 28℃. Strain H1 was subsequently maintained on medium â…  slants and stored as 30% (v/v) glycerol suspensions at -80℃.

2.2 Phenotypic characteristics
Strain morphology was inspect by light microscopy (Olympus microscope BX41) and Scanning Electron Microscope (Hitachi model S-3400N). Determination of the Gram type using the standard Gram reaction and the KOH lysis test method (Oren et al., 1997), after incubation for 48 h on medium Ⅰ at 28℃. Degradation of gelatin, starch, and Catalase activity, oxidase activity et al were determined based on the protocols 'Research on soil microorganisms' (Institute of soil science, Chinese academy of sciences, and micro-organism lab, 1985, Science Press, pp 41-120).

2.3 Experimental bacterial resistance
The strain SA-3 was used in the analysis of resilience. The bacteria were grown in liquid medium until OD600≈0.6, and then diluted 10-1~10-5. 3.5 microl liters of each dilution were inoculated to solid media â…¡ (LB supplemented with 0.5 mol NaCl ) and the LB medium supplemented with different concentrations of Na2CO3、NaCl and varied pH. The bacteria were grown at 28℃ for 48 h with the monitor of the growth. In E.coli JM109 as a control. In addition to NaCl resistance experiments outside the medium pH adjustment 7.0, The remaining conditions are the same as above.

2.4 Phylogenetic analysis of 16S rRNA gene sequences
Bacterium total genomic DNA was extracted and purified from bacteria according to the methods of  Griffiths (2000). The 16S rRNA gene fragments were cloned as described by Shigematsu (Shigematsu et al., 2003). The sequence of forward universal primers used in PCR amplification was 27F (5`-AGAGTTTGATCCTGGCTCAG-3`) and reverse primer was 1401R (5`-CGGTGTGTACAAGGCCC-3`). The reactions as followed: 95℃ for 2 min ; 35 cycles of 94℃ for 30 s, 54℃ for 40 s, and 72℃ for 2 min; 72℃ for 10 min. The 16S rRNA fragment was cloned into pMD18-Tvector (Takara, Biotechnology Co., Ltd) and transformed to competent cell JM109. The positive clones were identified by enzymatic digestion with Hindâ…¢ and BamHâ…  and confirmed by PCR. The cloned 16S rRNA gene was sequenced. The 16S rRNA sequences were analysed by using BLAST program and Clustal X program (Thompson et al., 1997). 16S rRNA phylogenetic tree was created by using MEGA program version 3.1 (Saitou and Nei, 1987).

Authors’ contributions
WS designed and conducted this experiment; TT participated the experiment design and data analysis; SKL is the person who takes charge of this project, including experiment design, data analysis, writing and modifying of the manuscript. All authors have read and approved the final manuscript.

Acknowledgements
Authors appreciate two anonymous reviewers for their useful critical comments and revising advice to this paper. And also we mentioned some reagent suppliers and sequencing service providers in this work, that doesn’t mean we would like to recommend or endorse their products and services.

References
Griffiths R.I., Whiteley A.S., O'Donnell A.G., and Bailey M.J., 2000, Rapid method for coextraction of DNA and RNA from natural environments for analysis of ribosomal DNA- and rRNA-based microbial community composition. Appl. Environ. Microbiol., 66(12): 5488-5491 doi:10.1128/AEM.66.12.5488-5491.2000 PMid:11097934    PMCid:92488

Luo H.Y., Wang Y.R., Miao L.H. Yang P.L., Shi P.J., Fang C.X., Yao B., and Fan Y.L., 2009, Nesterenkonia alba sp. nov., an alkaliphilic actinobacterium isolated from the black liquor treatment system of a cotton pulp mill, Int. J. Syst. Evol. Microbiol., 59(4): 863-868 doi:10.1099/ijs.0.003376-0 PMid:19329621

Oren A., Ventosa A., and Grant W.D., 1997, Proposed minimal standards for description of New Taxa in the order halobacteriales, International Journal of Systematic Evolutionary Microbiology, 47(1): 233-238

Saitou N., and Nei M., 1987, The neighbor-joining method: a new method for reconstructing phylogenetic trees, Mol. Biol. Evol., 4(4): 406-425 PMid:3447015

Shigematsu T., Tang Y.Q., Kawaguchi H., Ninomiya K., Kijima J., Kobayashi T., Morimura S., and Kida K., 2003, Effect of dilution rate on structure of a mesophilic acetate-degrading methanogenic community during continuous cultivation, J. Biosci. Bioeng., 96(6): 547-558 doi:10.1016/S1389-1723(04)70148-6 doi:10.1263/jbb.96.547

Stackebrandt E., Koch C., Gvozdiak O., and Schumann P., 1995, Taxonomic dissection of the genus Micrococcus: Kocuria gen. nov., Nesterenkonia gen. nov., Kytococcus gen. nov., Dermacoccus gen. nov., and Micrococcus Cohn 1872 gen. emend. Int. J. Syst. Evol. Microbiol., 45(4): 682-692

Thompson J.D., Gibson T.J., Plewniak F., Jeanmougin F., and Higgins D.G., 1997, The CLUSTAL_X windows interface: Flexible strategies for multiple sequence alignment aided by quality analysis tools, Nucleic Acids Res., 25(24): 4876-4882 doi:10.1093/nar/25.24.4876 PMid:9396791 PMCid:147148

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