Nanoscale 2012, 4:4712–4718.VE-822 chemical structure CrossRef BMN 673 supplier 16. Alexander KD, Skinner K, Zhang S, Wei H, Lopez R: Tunable SERS in gold nanorod dimers through strain control on an elastomeric substrate. Nano Lett 2010, 10:4488–4493.CrossRef 17. Zhang X-Y, Hu A, Zhang T, Lei W, Xue X-J, Zhou Y, Duley WW: Self-assembly of large-scale and ultrathin silver nanoplate films with tunable

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NC, Haynes CL, Oh S-H: Vertically oriented sub-10-nm plasmonic nanogap arrays. Nano Lett 2010, 10:2231–2236.CrossRef 26. Diebold ED, Mack NH, Doom SK, Mazur E: Femtosecond laser-nanostructured substrates for surface-enhanced Raman scattering. Langmuir 2009, 25:1790–1794.CrossRef 27. Lin C-H, Jiang L, Chai Y-H, Xiao H, Chen S-J, Tsai H-L: One-step fabrication of nanostructures by femtosecond laser for surface-enhanced Raman scattering. Opt Express 2009, 17:21581–21589.CrossRef 28. Jiang L, Ying D, Li X, Lu Y: Two-step femtosecond laser pulse train fabrication of nanostructured substrates for highly surface-enhanced Raman scattering. Opt Lett 2012, 37:3648–3650.CrossRef 29. Wang C, Chang Y-C, Yao J, Luo C, Yin S, Ruffin P, Brantley C, Edwards E: Surface enhanced Raman spectroscopy by interfered femtosecond laser created nanostructures. Appl Phys Lett 2012, 100:023107.CrossRef 30.

PubMedCrossRef 30. Cheng J, Randall AZ, Sweredoski MJ, Baldi P: SCRATCH: a protein structure and structural feature prediction server. Nucleic Acids Res 2005, (33 Web Server):W72–76. 31. Montgomerie S, Cruz JA, Shrivastava S, Arndt D, Berjanskii M, Wishart DS: PROTEUS2: a web server for comprehensive protein structure prediction and structure-based BMS345541 nmr annotation. Nucleic Acids Res 2008, (36 Web Server):W202–209. 32. SU5402 Enkhbayar P, Kamiya M, Osaki M, Matsumoto T, Matsushima N: Structural principles of leucine-rich repeat (LRR) proteins. Proteins 2004,54(3):394–403.PubMedCrossRef

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R: The architecture of parallel beta-helices and related folds. Prog Biophys Mol Biol 2001,77(2):111–175.PubMedCrossRef 35. Kobe B, Kajava AV: When protein folding is simplified to protein coiling: the continuum of solenoid protein structures. Trends Biochem Sci 2000,25(10):509–515.PubMedCrossRef 36. Baumann U: Crystal structure of the 50 kDa metallo protease from Serratia marcescens. J Mol Biol 1994,242(3):244–251.PubMedCrossRef 37. Kim HM, Park BS, Kim JI, Kim SE, Lee J, Oh SC, Enkhbayar P, Matsushima N, Lee H, Yoo OJ, et al.: Crystal structure of the TLR4-MD-2 complex with bound endotoxin antagonist Eritoran. Cell 2007,130(5):906–917.PubMedCrossRef 38. Jin MS, Kim SE, Heo JY, Lee ME, Kim HM, Paik SG, Lee H, Lee JO: Crystal structure of the TLR1-TLR2 heterodimer induced by binding of a tri-acylated lipopeptide. Cell 2007,130(6):1071–1082.PubMedCrossRef 39. Bendtsen JD, Nielsen H, von Heijne G, Brunak STA-9090 datasheet S: Improved prediction of signal peptides: SignalP 3.0. J Mol Biol 2004,340(4):783–795.PubMedCrossRef Authors’ contributions NM (corresponding author) carried out the molecular genetic

studies, participated in the sequence alignment and drafted the manuscript. HM performed dot plot analysis and radar chart analysis. TM contributed to the data analysis including the sequence alignment. KY conceived of the study, and participated in its design and coordination. All authors read and approved the final manuscript.”
“Background Escherichia coli typically colonize the mammalian and avian gastrointestinal tract and Farnesyltransferase other mucosal surfaces. While many of these strains are commensal, certain pathogenic strains have the ability to cause severe diseases [1]. Extraintestinal pathogenic E. coli (ExPEC) are a group of strains that are implicated in a large range of infections in humans and animals, such as neonatal meningitis, urinary tract infection, intra abdominal infection, pneumonia, osteomyelitis and septicaemia [2–4]. Among the typical extraintestinal infections caused by ExPEC in humans are urinary tract infections (UTIs), which are a major public health concern in developed countries costing healthcare systems billions of dollars annually [5].

HQ599507 Necrostatin-1 solubility dmso (V. cholerae 1383), HQ599508 (V. cholerae 7452), HQ599509 (V. cholerae 547), HQ599510 (V. cholerae 582), and HQ599511 (V. cholerae 175). Results V. cholerae strains from 2006 show reduced resistance profile compared to previous epidemic strains We analyzed

two V. cholerae O1 El Tor clinical strains, VC175 and VC189 (Table 1), isolated at the Luanda Central Hospital (Angola). These strains were collected during the peak (May) of the cholera outbreak reported in Angola in 2006. The two strains were sensitive to tetracycline, chloramphenicol, and kanamycin but VX-680 supplier showed a multiresistant profile to ampicillin, penicillin, streptomycin, trimethoprim, and sulfamethoxazole (see Table 1 for complete phenotype and genotype). Despite this significant multidrug resistance, these strains showed a narrower resistance profile compared to those isolated in the previous 1987-1993 cholera epidemic, which were also resistant to tetracycline, chloramphenicol, spectinomycin and kanamycin [11]. We found no evidence

for the presence of conjugative plasmids or class 1 integrons in the 2006 strains analyzed (data not shown), which might explain their reduced drug resistance profile. Indeed, strains from 1987-1993 were associated with the conjugative plasmid p3iANG that holds genes encoding the resistance to tetracycline, chloramphenicol, kanamycin, and spectinomycin PRI-724 mouse [11]. ICEVchAng3 is a sibling of ICEVchInd5 We assessed the presence of SXT/R391 family ICEs since they are a major cause of antibiotic

resistance spread among V. cholerae strains. Both strains were int SXT +, were shown to contain an ICE integrated into the prfC gene, and contained the conserved genes traI, traC and setR, respectively encoding a putative relaxase, a putative conjugation coupling protein, and a transcriptional repressor found in all SXT/R391 family members [31]. Based on these results we included this ICE in the SXT/R391 family and named it ICEVchAng3 according to the accepted nomenclature [32]. SXT/R391 ICEs exhibit significant genetic polymorphisms in hotspot content [12]. We used a first set of primers (primer set A), designed to PJ34 HCl discriminate between SXTMO10 and R391 specific sequences [25], in order to prove the identity of the ICE circulating in the 2006 Angolan strains. Genes floR, strA, strB, sul2, dfrA18, dfrA1, the rumAB operon, and Hotspots or Variable Regions s026/traI, s043/traL, traA/s054, s073/traF and traG/eex were screened. The 2006 strains exhibited the same SXTMO10/R391 hybrid ICE pattern. Intergenic regions traG/eex (Variable Region 4) and traA/s054 (Hotspot 2) showed the molecular arrangement described in SXTMO10, whereas region s043/traL (Hotspot 1) was organized as in R391. Variable Region 3, inserted into the rumB locus, contained genes that mediate resistance to chloramphenicol, streptomycin and sulfamethoxazole: floR, strA, strB, sul2.

jesenskae has at least two copies each of TOXD, TOXF, and TOXG. These three genes are 81-86% (nucleotide) and 81-85% (amino acid) identical to the corresponding genes in C. carbonum (Table 1).

Gene structures were experimentally verified by sequencing 5’ and 3’ RACE products. The intron/exon structures of all AjTOX2 genes are highly similar to C. carbonum (Figure 3). These three genes are clustered together on two distinct contigs in A. jesenskae (Figure 4). The arrangements of the genes within each contig are different in A. jesenskae and C. carbonum. In C. carbonum, TOXF and TOXG are clustered within ~300 bp (Figure 4), while at least 20 kb separates TOXD AC220 manufacturer from TOXF and TOXG in C. carbonum[9]. TOXD expression is regulated with the other genes of TOX2 by the transcription factor TOXE, but its disruption gave no detectable HC-toxin or virulence https://www.selleckchem.com/products/bix-01294.html phenotype (unpublished results from this lab). TOXF is required for HC-toxin production and is predicted to encode a member of the branched-chain amino acid aminotransferase family [23]. Although its precise biochemical function is unknown, a plausible function of TOXF is to aminate a precursor of Aeo, e.g., the fatty acid product of TOXC and TOXH. The function of TOXG has been established as an alanine racemase [24]. TOXG is a member of the pyridoxal-containing serine hydroxymethyl transferase

superfamily [25]. AjTOXE- HC-toxin-specific transcription factor TOXE encodes a transcription factor that regulates the known genes of TOX2 in C. carbonum[26, 27]. It contains a bZIP DNA binding click here domain at its N terminus and four ankyrin repeats at its C-terminus [27]. C. carbonum strain SB111 has two copies of TOXE, one clustered with the other TOX2 genes and one on a separate chromosome. In other strains, both copies of TOXE are Tolmetin on the same chromosome [9]. A. jesenskae also has two copies of AjTOXE on two separate contigs, but it is not known if these contigs are on the same or different chromosomes. Within A. jesenskae the two

copies of AjTOXE are 85% (nucleotide) and 76% (amino acid) identical (Table 1). This is a lower degree of identity than for any of the other copies of the AjTOX2 genes to each other. The two copies average 61% amino acid identity between C. carbonum and A. jesenskae (Table 1). This degree of conservation between TOXE and AjTOXE is lower than for any of the other TOX2 proteins (see Discussion). In C. carbonum, TOXE binds to promoters of the TOX2 genes containing the “Tox Box” motif, ATCTCNCGNA [27]. Analysis of the contigs containing the AjTOX2 genes indicates the probable presence of similar motifs in their putative promoter regions (data not shown). However, their location in relation to the genes themselves is unclear at this time, because the transcriptional start sites of the AjTOX2 genes have not been experimentally verified.

Med Microbiol Immunol 2009, 198:221–238.PubMedCrossRef 10. Kohler S, Foulongne V, Ouahrani-Bettache S, Bourg G, Teyssier J, Ramuz M, Liautard JP: The analysis of the intramacrophagic virulome of Brucella suis deciphers the environment encountered by the pathogen inside the macrophage host cell. Proc Natl Acad Sci USA 2002, 99:15711–15716.PubMedCrossRef 11. Volkert MR, Nguyen DC: Induction of specific Escherichia coli genes by sublethal treatments with alkylating agents. Proc Natl Acad Sci USA 1984, 81:4110–4114.PubMedCrossRef

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2010, 192:3235–3239.PubMedCrossRef 17. Hallez R, Mignolet J, Van Mullem V, Wery M, Arachidonate 15-lipoxygenase Vandenhaute J, Letesson JJ, Jacobs-Wagner C, De Bolle X: The asymmetric distribution of the essential histidine kinase PdhS indicates a differentiation event in Brucella abortus . EMBO J 2007, 26:1444–1455.PubMedCrossRef 18. Bowles T, Metz AH, O’Quin J, Wawrzak Z, Eichman BF: Structure and DNA binding of alkylation response protein AidB. Proc Natl Acad Sci USA 2008, 105:15299–15304.PubMedCrossRef 19. Rippa V, Amoresano A, Esposito C, Landini P, Volkert M, Duilio A: Specific DNA binding and regulation of its own expression by the AidB protein in Escherichia coli . J Bacteriol 2010, 192:6136–6142.PubMedCrossRef 20. Sedgwick B: Repairing DNA-methylation damage. Nat Rev Mol Cell Biol 2004, 5:148–157.PubMedCrossRef 21. Volkert MR: Adaptive response of Escherichia coli to alkylation damage. Environ Mol Mutagen 1988, 11:241–255.PubMedCrossRef 22. Lawley PD, Brookes P: Cytotoxicity of alkylating agents towards sensitive and resistant strains of Escherichia coli in relation to extent and mode of alkylation of cellular macromolecules and repair of alkylation lesions in deoxyribonucleic acids. Biochem J 1968, 109:433–447.PubMed 23. Alvarez G, Campoy S, Spricigo DA, Teixido L, Cortes P, Barbe J: Relevance of DNA alkylation damage repair systems in Salmonella enterica virulence. J Bacteriol 2010, 192:2006–2008.PubMedCrossRef 24.

Greeley J, Stephenes IE, Bondarenko AS, Johansson TP, Hansen HA, Jaramillo TF, Rossmeisl J, Chorkendorff I, Nørskov JK: Alloy of platinum and early

transition metals as oxygen reduction electrocatalysts. Nat Chem 2009, 1:552–556. 10.1038/nchem.367CrossRef 17. Sepa DB, Vojnovic MV, Damjanovic A: Reaction intermediates as a controlling factor in the kinetics and mechanism of oxygen reduction at platinum electrodes. Electrochim Acta 1981, 26:781–793. 10.1016/0013-4686(81)90037-2CrossRef 18. Garsany Y, Barurina OA, Swider-Lyons KE, Kocha SS: Experimental methods for quantifying the activity of platinum electrocatalysts for the oxygen reduction reaction. Anal Chem 2010, 82:6321–6328. 10.1021/ac100306cCrossRef 19. Guo S, Sun S: FePt nanoparticles assembled on graphene as enhanced Selleck Barasertib catalyst for oxygen reduction reaction. J Am Chem Soc 2012, 134:2492–2495. 10.1021/ja2104334CrossRef 20. Yung TY, Lee JY, Liu LK: Nanocomposite for methanol: synthesis and characterization of cubic Pt nanoparticles on graphene sheets. Sci Technol Adv Mater 2013, 14:035001. 10.1088/1468-6996/14/3/035001CrossRef 21. Wu J, Zhang J, Peng Z, Yang S, ITF2357 clinical trial Wangner FT, Yang H: Truncated octahedral Pt 3 Ni oxygen reduction reaction electrocatalysts. J Am Chem Soc 2010, 132:4984–4985. 10.1021/ja100571hCrossRef 22. Wang Caspase inhibitor clinical trial Y, Wang S, Xiao M, Han D, Hickner M, Meng Y: Layer-by-layer self-assembly of PDDA/PSS-SPFEK composite

membrane with low vanadium permeability for vanadium redox flow battery. RSC Adv 2013, 35:15467–15474.CrossRef 23. Wang S, Wang X, Jiang SP: Self-assembly of mixed Pt and Au nanoparticles on PDDA-functionalized graphene as effective electrocatalysts for formic acid oxidation fuel cells. Phys Chem Chem Phys 2011, 13:6883–6891. 10.1039/c0cp02495cCrossRef 24. Wang S, Yu D, Dai L, Chang JB: Polyelectrolyte-functionalized graphene as metal-free electrocatalysts for oxygen reduction. ACS Nano 2011, 5:6202–6209. 10.1021/nn200879hCrossRef 25. Yuan L, He Y: Effect of surface charge of PDDA-protected gold nanoparticles on the specificity and efficiency of

DNA polymerase chain reaction. Analyst C1GALT1 2012, 138:539–545.CrossRef 26. Zhu LP, Liao GH, Xiao HM, Wang JF, Fu SY: Self-assembled 3D flower-like hierarchical β-Ni(OH) 2 hollow architectures and their in situ thermal conversion to NiO. Nanoscale Res Lett 2009, 4:550–557. 10.1007/s11671-009-9279-9CrossRef 27. Wang H, Kou X, Zhang J, Li J: Large scale synthesis and characterization of Ni nanoparticles by solution reaction method. Bull Mater Sci 2008, 31:97–100. 10.1007/s12034-008-0017-1CrossRef Competing interests The authors declare that they have no competing interests. Authors’ contributions TYY, LYH, and TYL conceived and designed the experiments. PTC, LYH, TYC, and KSW performed the experiments. TYY, LYH, TYC, CYC, and KSW contributed ideas and material analyses. TYY, TYL, and LKL wrote the manuscript. This work was performed under the supervision of LKL. All authors read and approved the final manuscript.

The electroporated cells were diluted in 1 ml LB and incubated at 37°C for three hours. The transformants were then selected on the antibiotic-imbued plates. Scarless gene modification in P. aeruginosa Scarless gene modification strategy was described in Fig. 2. First the sacB-bla cassettes were amplified from plasmid pEX18Ap with the primers F1 and R1 [16]. The numbers of primers corresponded to the steps of PCR amplification. The electro-transformation of the sacB-bla cassette into the PAO1/pRKaraRed

competent cells was performed as described above. Transformants were screened on LB LY2874455 mouse plates supplemented with 500 μg/ml carbenicillin and 50 μg/ml tetracyclin. The colonies with CarbRTetR Geneticin clinical trial phenotypes confirmed by PCR detection and DNA sequencing were regarded as positive clones. Next, the sacB-bla removal cassettes were amplified from the genomic DNA of the first-step strain with the primers F2 and R2. Then this fragment was electro-transformed into the competent cells of the first-step to perform the second recombination. Electro-transformed cells were spread on LB plates supplemented with 10% sucrose and 50 μg/ml tetracycline. The transformants were further selected parallel on the LB plates Quisinostat cost with 10% sucrose and 50 μg/ml tetracycline, and the LB plates with 500 μg/ml carbenicillin and 50 μg/ml tetracycline.

The colonies with SucRCarbS phenotypes confirmed by PCR detection and DNA sequencing were regarded as positive recombinants. Twelve genes, two large operons and one nucleotide site were selected as target and their primers for PCR amplification were listed in Additional file 1, Table S1. System efficiency analysis The influences of L-arabinose concentration, induction time and the length of homology region on

the efficiency of homologous recombination were analyzed. phzS gene was selected as target. First, the PAO1/pRKaraRed cultures were induced with L-arabinose of different concentrations (ranging from 0.05% to 1.0%) for three hours. Then the PAO1/pRKaraRed cultures were Buspirone HCl induced with L-arabinose of suitable concentration for different time (from 1 h to 12 h). Finally, the PCR products with homology regions of different lengths (50 bp, 60 bp, 100 bp) were used to perform homologous recombination. Control experiments and screen procedures were set same as described above. The efficiencies of recombination were calculated by dividing the number of positive colonies with the number of growing colonies. Construction of three-gene deleted strain PCA and HPLC analysis of phenazine derivatives Sequential gene modifications of multiple target genes were achieved by several rounds of recombination steps. The recombination efficiency was also detected using phenotype screen, PCR detection and DNA sequencing. The strain with three-gene deletions (PAO1, ΔphzHΔphzMΔphzS) was named as PCA.

For the quantum transport, we use the non-equilibrium Green’s function formalism [14]. We GANT61 manufacturer consider the coherent limit where it is equivalent to the Landaüer’s approach, and the current can be evaluated from the transmission as below: (2) where transmission is T(E) = tr(Γ s GΓ d G +). The Green’s function for the channel is (3) where I is an identity matrix and U L is the Laplace’s potential drop. Self-energies Dibutyryl-cAMP and broadening functions are and Γ s,d

= i[Σ s,d − Σ s,d +], respectively. are the contact Fermi functions. μ s,d are source/drain chemical potentials. μ d is shifted due to drain bias as μ d = μ o − qV d and μ s = μ o, where μ o is the equilibrium chemical potential. Results and discussion We next discuss the numerical results for a transistor with α = 0.4 and BWo = 0.1 eV. The transfer characteristics with V d = 0.16, 0.18 and 0.2 V are shown in Figure 2a. A steep subthreshold slope is obtained with a high on/off current ratio. The threshold voltage depends on the drain voltage V d, and it increases with the drain bias – a trend opposite to the drain-induced barrier

lowering of a FET. The subthreshold current much below the threshold voltage, which is due to the reflections from the barrier of the near-midgap state, decreases exponentially. Figure 2 Transport characteristics. (a) Transfer characteristics show steep subthreshold characteristics with drain-voltage dependent threshold voltage shift. (b) Output characteristics show a saturating behavior followed by a negative GM6001 concentration differential resistance. (c) With increasing drain bias, the Adenosine triphosphate transmission window shrinks due to a spectral misalignment (Addition file 1). (d) The increasing Fermi function difference between the two

contacts and the decreasing transmission lead to an increasing and then decreasing T(E)[f s − f d] function. We further report the output characteristics in Figure 2b for V g = 0.04, 0.08, 0.12, 0.16, and 0.2 V, which show a negative differential resistance (NDR) behavior that is crucial for the low-power inverter operation (Additional file 1). The current cut-off mechanism is similar to the Bloch condition through minibands in superlattices, giving rise to an NDR event, when the drain voltage exceeds the miniband width [15, 16]. The miniband in superlattices is formed by the overlap of quantized states through tunnel barriers, inherently leading to small miniband widths and large effective masses [17]. The NDR events mediated by minibands have been reported in III-V heterostructures [18] and graphene superlattices [19]. However, the peak-to-valley ratio in such structures is limited to about 1.1 to 1.2. In comparison, the NDR feature reported for near-midgap state in this work shows a peak-to-valley ratio of greater than 103, which is important for the low-power operation.

The selected strains were isolated from blood (n = 11), CSF (n = 3) and other sterile fluids (n = 3); c) Forty-six pneumococci were selected from nasopharyngeal carriers aged from 1 to 4 years old, in Oviedo (Northern

Spain) in 2004–2005 [23] (Additional file 1). These strains were representative of 29 dominant PFGE patterns found among 365 pneumococci isolated from children attending 23 Sapanisertib day-care centers. Antimicrobial susceptibility testing The minimal inhibitory concentration (MIC) was determined by microdilution following CLSI guidelines [26] using a panel of antimicrobials which included penicillin, erythromycin, clindamycin, tetracycline, chloramphenicol and cotrimoxazol. Resistant strains were defined according to CLSI criteria [27]. S. pneumoniae ATCC 49619 was used as control. Multilocus sequence typing (MLST) and eBURST MLST was performed as described previously [28]. The allele’s number and sequence types (ST) were ��-Nicotinamide assigned using the pneumococcal MLST website [29]. Lineage assignment was achieved by eBURST analysis [30, 31]. PspA detection The PCRs were carried out in a standard PCR mixture of 50 μl containing 2.5 mM of MgCl2, 240 μM (each) of deoxynucleoside triphosphates (dNTPs), 0.3 μM of each primer, and 2 U of Taq DNA polymerase (AmpliTaq Gold®, Roche). The cycle

conditions consisted of: an initial 94°C (10 min), 30 cycles of 94°C (1 min), 55°C (1 min) and 72°C (3 min), followed by 72°C (10 min). A multiplex PCR reaction was tested [32], but some samples did not amplify with LSM12/SKH63 [32, 33] or LSM12/SKH52 [22] primer combinations. The combination of LSM12/SKH2 Selleckchem S3I-201 primers [16] was successfully used for all samples except one. The isolate that did not amplify was retested with the same cycle pattern at an annealing temperature of 52°C and with different primer combinations (LSM12/SKH63, LSM12/SKH52 and LSM12/SKH2). Controls

for PspA family 1 (Spain14-ST18) and PspA family 2 (Spain23F-ST81) were run in each reaction set. PCR products were purified and sequenced Alectinib using SKH2 primer, as described elsewhere [34]. Sequence edition was performed using the SeqScape version 2.1.1 (Applied Biosystems) software, while DNA sequences were assigned using BLAST [35]. Clade type was established when the closest match presented identity higher than 95% (Figure 1). The phylogenetic and molecular evolutionary analyses were conducted using MEGA4 version 4.1 software [36]. The evolutionary history was inferred using the Neighbor-Joining method and the bootstrap consensus tree inferred from 1000 replicates. The evolutionary distances were computed using the Kimura 2-parameter method [36]. Figure 1 Phylogenetic tree of a 373-bp region that includes psp A clade-defining region. Phylogenetic and molecular evolutionary analyses were conducted with the MEGA4 program (version 4.1) [36] by the Neighbor-Joining method. Only bootstrap confidence intervals exceeding 90% are shown.

-terminus of the primer. The GC-clamp sequence was CCCCGTGCTCCCCCGCCAATTCCT;. DNA extraction DNA extraction for the rumen epithelium

(0.1 g wet click here weight) samples was conducted using a QIAamp® DNA Stool Mini Kit (QIAGEN, Hilden, Germany). Prior to extraction, the samples were pretreated Selleck Idasanutlin using the FastPrep®-24 Instrument (MP Biomedicals, South Florida, USA). Then, the procedure followed the kit instructions. DNA extraction for the culture supernatant (5 ml), the rumen fluid (3 ml), and the solid samples (0.3 g wet weight) were conducted using the cetyltrimethylammonium bromide method [32]. Prior to extraction, all the samples were washed two or three times with PBS buffer.

The DNA extracts were dissolved in 100 μl TE buffer and DNA yield was quantified using a NanoDrop ND-1000 Spectrophotometer (Nyxor Biotech, Paris, France). The DNA extracts were diluted in ddH2O prior to PCR reactions and 1 μl of the diluted DNA solutions (c.10 ~ 20 ng) GSK2118436 research buy were used as templates. PCR-DGGE analysis of methanogen community in subcultures of the co-culture with anaerobic fungi PCR-DGGE analysis of the methanogen community in co-culture with anaerobic fungi was conducted with primers 519f/915GCr (Table 3) according to the methods described in our previous study [12]. The PCR reaction system (50 μl) contained 0.2 μM

of both primers, 240 μM of each dNTP, 1.5 mM of MgCl2 and 2.5 units of Taq DNA polymerase, 1 μl of template DNA. The amplification parameters were as follows: initial RVX-208 denaturation at 94°C for 4 min, then 35 cycles of 94°C for 30 s, 57°C for 40 s and 72°C for 40 s, and last extension at 72°C for 10 min. DGGE was performed using a Dcode DGGE system (Bio-Rad, Hercules, USA) with 6% (w/v) polyacrylamide gels (acrylamide/N, N’-methylene bisacrylamide ratio, 37: 1 [w/w]) in 0.5 × TAE buffer. The denaturant gradient range of the gel was from 35% to 75%, in which 100% denaturant contained 7 mol · L−1 urea and 40% (v/v) formamide. The electrophoresis was initiated by pre-running for 10 min at 200 V and subsequently ran at 85 V for 16 h at 60°C. The gel was stained with AgNO3 and scanned using GS-800 scanner (Bio-Rad, Hercules, USA). The DGGE profile was analysed by Molecular Analyst 1.61 software (Bio-Rad, Hercules, USA). DGGE bands were excised from the gel and rinsed with ddH2O. The DNA of each band was eluted in sterile TE buffer by incubation for 12 h at 37°C, and served as the template for re-amplification with primers 519f/915r. The PCR products of re-amplification were cloned in Escherichia coli Top10 by using the pGEM-T Easy Vector System (Promega, Madison, WI, USA).