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Using Polymerase Chain Reaction (PCR) Melting Profile and Protein Fingerprint Profile Analyses to Identify Methanothermobacter Marburgensis DX01

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Using Polymerase Chain Reaction (PCR) Melting Profile and Protein Fingerprint Profile Analyses to Identify Methanothermobacter Marburgensis DX01 African Journal of Microbiology Research Vol. 5(26) pp. 4555-4561, 16 November, 2011 Available online at http://www.academicjournals.org/AJMR ISSN 1996-0808 ©2011 Academic Journals DOI: 10.5897/AJMR11.586 Full Length Research Paper Using polymerase chain reaction (PCR) melting profile and protein fingerprint profile analyses to identify Methanothermobacter marburgensis DX01 Xia Ding1*, Xiao-Jue Peng1, Wen-Zhuo Li1, Yao-Hui Cai2 and Wei-Jun Yang3 1College of Life Sciences, Nanchang University, Nanchang, Jiangxi, 330031, China. 2Jiangxi Academy of Agricultural Sciences, Nanchang, Jiangxi, 330000, China. 3College of Life Sciences, Zhejiang University, Hangzhou, Zhejiang, 310058, China. Accepted 17 October, 2011 A thermophilic new strain of Methanothermobacter marburgensis DX01, with a different morphology from M. thermautotrophicus delta H, was isolated from a hot spring in China. To provide further molecular evidence to identify the methanogen M. marburgensis DX01, polymerase chain reaction melting profile (PCR MP) technique and protein fingerprint profiling were performed. Both DNA and protein fingerprint profiles showed significant differences between M. marburgensis DX01 and M. thermautotrophicus delta H. In addition, five interesting DNA fragments obtained from PCR MP of the DX01 strain were excised and sequenced. We found that all five DNA fragments were unique to M. marburgensis DX01. Expression of induced proteins in M. marburgensis DX01 was analyzed by protein fingerprint profiling and a protein that was more highly expressed at 70°C was excised and identified by N-terminal amino acid sequencing. We found that the up-regulated protein was Methyl-coenzyme M reductase І, which was 97.6% amino acid sequence identity with that of M. thermautotrophicus delta H. These studies may provide clues for a better understanding of the new isolate of thermophilic M. marburgensis. Key words: Methanothermobacter, polymerase chain reaction (PCR) melting profile, protein fingerprint profile analyses. INTRODUCTION There are a large number of extremely thermophilic anaerobic digesters, digested sludge and hot springs. organisms in the archaeal domain (de Poorter et al., Although these strains were assigned to a single species, 2007; Schill et al., 1996). The thermophilic species of there are still certain differences between the strains. Methanothermobacter are able to produce their energy With the development of new taxonomy techniques, from the oxidation of H2 and reduction of CO2 to methane various taxonomic subdivisions of Methanothermobacter (Valentine, 2007). Growth of Methanothermobacter is into separate species have been proposed. In 2001, characterized by a requirement for gaseous energy and Methanothermobacter was redefined as carbon sources and low biomass. In addition, Methanothermobacter defluvii, Methanothermobacter Methanothermobacter is thermophilic and strictly marburgensis, Methanothermobacter thermautotrophicus, anaerobic (Farhoud et al., 2005; Schill et al., 1996). Methanothermobacter thermoflexus, Currently, over ten Methanothermobacter strains have Methanothermobacter thermophilus and been found in a number of environments, including Methanothermobacter wolfeii, as cited in the second edition of the Bergey’s Manual of Systematic Bacteriology and published in the validation list no. 85. Over the past 20 years, a large number of DNA-based *Corresponding author. E-mail:[email protected]. Tel: +86 techniques have been introduced into the field of 791 3969163. Fax: +86 7913. microbial characterization and taxonomy. These include 4556 Afr. J. Microbiol. Res. restriction endonuclease analysis, molecular NiCl2∙6H2O 0.025 g; NaSeO3∙6 H2O 0.0003 g). After adjustment to hybridization, and PCR amplification; most of these pH 7.0, the medium was prepared and sterilized under a strictly anaerobic H2 and CO2 atmosphere (80:20). techniques use PCR for detection of fragments (Dnyaneshwar et al., 2006; Frias-Lopez et al., 2008; Krawczyk et al., 2006; Lopez-Ribot et al., 2000; Pandya Electron microscopy (EM) at different growth temperature et al., 2009). These genomic fingerprinting methods were Strains were grown to late exponential phase at different incubation developed to detect DNA sequence polymorphisms by temperatures (50, 60, 65 and 70°C). The electronic graph was using general principles such as restriction fragment performed using electron microscopy (EM). length polymorphisms (RFLPs) (Botstein et al., 1980), random amplification of polymorphic DNA (RAPD) (Williams et al., 1990), or repetitive element PCR (rep- DNA extraction PCR) (Georghiou et al., 1994). Chromosomal DNA was obtained using previously described Nowadays, physiological characterization, phylogenetic methods (Jarrell et al., 1992). Total DNA was quantified on a analysis of the 16S rRNA, DNA-DNA hybridization, Genova UV/visible spectrophotometer at 260 nm. analysis of the GC percentage, antigenic fingerprinting, and plasmid and phage typing have all been used for Ligation mediated PCR performed at low denaturation type strains of Methanothermobacter (Wasserfallen et al., temperatures (PCR melting profiles) 2000). The performance and convenience of the PCR melting profile (PCR MP) technique, which is based on The PCR melting profile (PCR MP) was performed according to using low denaturation temperatures during ligation previously described methods (Masny and Plucienniczak, 2003). mediated PCR (LM PCR) of bacterial DNA, demonstrates Briefly, genomic DNA was digested with HindIII. Digested DNA was ligated with two oligonucleotides forming an adaptor: POWIE 5'- that PCR MP is a rapid method that offers good CTCACTCTCACCAACGTCGAC-3' and HINLIG 5'- discriminatory power (Krawczyk et al., 2009; Krawczyk et AGCTGTCGACGTTGG-3'. The ligation products were used in the al., 2007; Krawczyk et al., 2006; Masny and next round of PCR as an amplification template with primer Plucienniczak, 2003; Mueller et al., 2001; Ngoc et al., POWAGCTT 5'-CTCACTCTCACCAACGTCGACAGCTT-3'. PCRs 2008). were performed by incubating the samples for 2 min at 72°C to A new thermophilic archaea M. marburgensis DX01 release unligated HINLIG oligonucleotides, followed by the addition of 1 µl of Taq DNA polymerase (2.5 U/μl). The reaction mixture was was isolated from a hot spring in China (Ding et al., incubated for an additional 5 min at 72°C to fill in the single- 2010). The isolate showed some distinct phenotypic stranded ends and create amplicons, after which the samples were characteristics in pellet color and morphology. Previously, initially denatured at (80 + x) °C for 1 min, followed by 21 cycles of M. marburgensis and M. thermautotrophicus were denaturation at (80 + x) °C for 40 s, with annealing and elongation thought to belong to the same species: at 72°C for 2 min (where x is a digit between 0 and 8, and is constant for a single PCR reaction). Methanobacterium thermoautotrophicum. Strain delta H was a type strain. The two species exhibited extremely similar physiological characteristics. However, DNA-DNA Comparison of PCR MPs of two strains hybridization studies revealed they lacked similarity in The products of PCR MPs were separated by 2% agarose gels. sequence (Wasserfallen et al., 2000). The differences Electrophoresis was performed in 1·Tris–acetate–EDTA buffer at 50 between the two species in genomic sequence and V. Gels were stained by ethidium bromide. Interesting DNA protein expression were still unknown. Additionally, it was fragments were recovered from the agarose gel using the Agarose not known whether the new DX10 strain possessed Gel DNA Fragment Recovery Kit (TaKaRa, Dalian, China). unique genes. In the present study, to provide further Recovered PCR product was ligated into the pUCm-T vector (TaKaRa, Dalian, China) and transformed into E. coli DH5α cells. molecular evidence of the difference between the strains, The cloning procedure was conducted according to the PCR MP and protein fingerprint profiles were used to manufacturer’s instructions. Sequencing reactions were run on an create a more complete picture of the variation in Applied Biosystems 3730 sequencer (Invitrogen, Shanhai, China). genomes and protein expression to fingerprint a novel methanogen strain DX01. N-terminal protein sequencing MATERIALS AND METHODS Purified proteins from the fractions were first separated by SDS- PAGE and then transferred via electroblot onto PVDF membranes. Culturing Protein bands on the PVDF membrane were visualized by staining with G-250 Coomassie stain solution, after which protein bands of Methanothermobacter strains were grown in anaerobic medium interest with the correct molecular weight were cleaved and the N- using the Hungate technique (Balch et al., 1979). The culture terminal amino acid sequence was determined on the Procise- -1 medium contained (L ): MgCl2·6H2O, 0.1 g; NaCl 0.6 g; K2HPO4, Protein Sequencing System (Applied Biosystems) using the 1.5 g; NH4Cl, 1.5 g; sodium cysteine 0.5 g; NaHCO3, 1.0 g; sodium manufacturer’s pulse-liquid Edman degradation chemistry cycles. resazurin 0.001 g; trace element solution 10 ml (MgSO ∙7H O 3 g; 4 2 MnSO4∙4H2O 0.5 g; NaCl 10 g; FeSO4∙7H2O 0.1 g; CoCl2∙6H2O 0.1 Cloning and nucleotide sequencing g; CaCl2∙2H2O 0.1 g; ZnSO4∙7H2O 0.1 g; CuSO4 0.01 g; KAl(SO4)2∙12H2O 0.01 g; H3BO3 0.01 g; Na2MoO4∙2H2O 0.01 g; Degenerate primers (5'-ATG ATA TGG CAC AAT ACT A-3' and 5'- Ding et al. 4557 Figure 1. Morphology of M. marburgensis DX01. (a) Color of the cell pellet. (b) Electron microscopy of cells grown at 50°C (A, E), 60°C (B, F), 65°C
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