PCR-generated artefact from 16S rRNA gene-specific primers.

Department of Microbiology and Immunology, University of Melbourne, Parkville, Victoria 3010, Australia.
FEMS Microbiology Letters (Impact Factor: 2.05). 08/2005; 248(2):183-7. DOI: 10.1016/j.femsle.2005.05.043
Source: PubMed

ABSTRACT Artefacts consisting of concatenated oligonucleotide primer sequences were generated during sub-optimally performing polymerase chain reaction amplification of bacterial 16S rRNA genes using a commonly employed primer pair. These artefacts were observed during amplification for terminal restriction fragment length polymorphism analyses of complex microbial communities, and after amplification from DNA from a microbial culture. Similar repetitive motifs were found in gene sequences deposited in GenBank. The artefact can be avoided by using different primers for the amplification reaction.

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    Dataset: wang-12
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    ABSTRACT: Background and Aims Plant growth-promoting bacteria, mainly diazotrophs and phosphate solubilizers, can reduce the use of chemical fertilizers for rice crops. Here, diazotrophic bacteria isolated from rice were screened for their ability to solubilize inorganic P (Pi) in vitro and in association with rice plants cultivated in pots. Methods Forty-nine isolates were tested for the ability to solubilize Pi on NBRIP and GL agar plate media and seven selected strains were further evaluated in NBRIP liquid medium. Three of these strains were inoculated in rice plants grown in soil pots containing 15N-labeled fertilizer and two sources of P: tricalcium phosphate (TCP) or simple superphosphate (SSP). The dry matter, yield, N, P, and the 15N content accumulated in plant tissues were measured at 135 days after planting. Results Seven strains belonging to the genera Herbaspirillum and Burkholderia formed a halo of solubilized Pi on agar plates. The Burkholderia strains showed peak soluble P (around 200 mg P L−1) on the fifth day when grown in NBRIP liquid medium for 14 days. Inoculation of Herbaspirillum strains (H18, ZA15) and a Burkholderia vietaminensis strain (AR114) increased rice grain yield from 33 to 47 % with TCP and 18 to 44 % with TSS, respectively. The bacterial inoculation led to enhanced N-use efficiency of the 15N-labeled fertilizer. Conclusion These results suggest that the selection and use of P-solubilizing diazotrophic bacteria are a good strategy to promote P solubilization and/or N use efficiency in rice plants.
    Plant and Soil 369(1-2). · 3.24 Impact Factor
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    ABSTRACT: Efficient alkaline protease producer was isolated from hyper alkaline-saline Lonar Soda Lake and identified as Bacillus alcalophilus LW8 using culture dependent techniques like morphological characters, microscopic features, biochemical pattern, physiological characters and molecular analysis. The 16S rRNA gene sequence was submitted in GenBank nucleotide repository under the accession number KC689353. Alkaline protease production was optimized by adopting one-variable-at-a-time approach using submerged fermentation system in modified fermentation medium (MFM). The optimized components of MFM were (w/w) casein (1%), sugarcane molasses (1%), NaCl (1%), ammonium sulphate (0.5%), KH2PO4 (0.05%), K2HPO4 (0.05%) and Na2CO3 (1%). The optimized culture conditions were used for alkaline protease production. Final yield of partially purified alkaline protease was achieved 53.35% after dialysis. The molecular weight of the dialyzed alkaline protease was determined by SDS–PAGE as 27 kDa. As per Lineweaver-Burk plot, calculated Km and Vmax values were 24 mg/mL and 1000 U/mg respectively. The enzyme was remarkably stable at the pH range of 7.0–12.0 with optimum activity at pH 10.0. LW8 alkaline protease was completely inhibited by PMSF at 10 mM concentration, indicating that the enzyme belongs to class serine protease. The metal ions viz. Ca2+, Ba2+, Mg2+, Zn2+, Fe3+, Cu2+, Mn2+ had increased catalytic activity of partially purified alkaline protease. Partially purified LW8 alkaline protease effectively decomposed gelatinous coating on X-ray film, hydrolyzed blood clot, removed blood stain from a piece of cotton fabric and hairs from a piece of goat skin.
    Journal of Taibah University for Science. 01/2014;


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May 21, 2014