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Molecular Soil Biology (online), 2011, Vol. 3 No.2, 5-8
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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 Ⅰ ( 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 Ⅰ slants and stored as 30% (v/v)
glycerol suspensions at -80℃ .
3.2 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).
3.3 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
Ⅱ (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.
3.4 Phylogenetic analysis of 16S rRNAgene 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`-AGAGTTTG
ATCCTGGCTCAG-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 HindⅢ and Bam HⅠ 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 MEGAprogram 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:10.11
28/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:939
6791 PMCid:147148