In addition, the qPCR assays were validated by testing the specificity of the primers on

the following 12 closely related species: Klebsiella pneumoniae, Klebsiella oxytoca, Acinetobacter calcoaceticuc, Burkholderia cepacia, Burkholderia sp., Ralstonia eutrophus. Brevundimonas sp., Stenotrophomonas maltophilia, Pseudomonas putida, Pseudomonas fluorescens, Pseudomonas aeruginosa and Pseudomonas stutzeri. The assays were specific for their targets and gave no or very high Ct values for the nontarget groups equal to the nontemplate control (data not shown), which furthermore confirmed the specificity of the primers. Pyrosequencing of PCR products amplified from the sludge soil sample with the Burkholderia primers resulted in Raf inhibitor 24 890 sequences longer than 250 bp. RDP classification of these sequences showed that 99% of the sequences belonged this website to Betaproteobacteria and of these only 8% to Burkholderia (Fig. 1).

Based on these results, the Burkholderia primer specificity is 8%. Because of the low primer specificity, no further data treatment was carried out. Pyrosequencing of PCR products amplified from the same soil sample with the Pseudomonas-specific primers generated a total of 24 354 sequences longer than 150 bp. RDP classification of these sequences showed that 98.76% belonged to Pseudomonas (Fig. 2), 0.56% to unclassified bacteria, 0.40% to unclassified Pseudomonadacea, and the last 0.28% belonged to closely related bacteria. Based on these numbers, we estimated that the Pseudomonas primers have the specificity close to 99%. Using the RDP Pyrosequencing Nintedanib (BIBF 1120) pipeline, the rarefaction curves estimated that 0.5 g of soil contains c. 200 different Pseudomonas OTUs at 3% maximum cluster distance (Fig. 3). To assess the distribution of the Pseudomonas community in soil, clusters containing more

than 50 identical copies were blasted against the full RDP database to identify the species level. In most cases, a high identity score on a single species was possible, but in a few blasts several species appeared with identical similarity scores. Where several hits were shown with identical similarity score, the number of sequences in the cluster was distributed evenly between the different hits. The different clusters and the number of species and sequences they represent are illustrated in Fig. 4. Using this method, the most dominant Pseudomonas groups in the soil are clearly uncultured Pseudomonas and P. putida followed by P. flourescens and Pseudomonas sp. The figure also shows that there is a rather diverse mixture of Pseudomonas species present in the soil. Pseudomonas was quantified in two different soils: one treated with household compost and the other with sewage sludge. The two assays, SYBR Green I and hydrolysis probes detection format, were validated and compared. All qPCR runs showed high efficiency c. 100% and R2-value in the average range between 0.981 and 0.999 (data not shown).

, 2003). Vemurafenib These ‘universal’ primers have been designed based on the conserved regions among most archaeal and/or bacterial 16S rRNA genes. It should be noted that the detectable members are constrained by the nucleotide sequence of the PCR primers used, meaning that these ‘universal’ primers may not be completely universal. Diverse Archaea have been detected in terrestrial and marine environments (Robertson et al., 2005; Schleper et al., 2005). Archaeal diversity in natural environments has often been investigated by PCR-based analysis using Arch21F as a forward primer as reported previously (Delong,

1992) or other primers (Massana et al., 1997; Dojka et al., 1998; Eder et al., 1999; Reysenbach et al., 2000). These primers were designed based on the conserved regions of archaeal 16S rRNA gene sequences between positions 7 and 26 in the Escherichia coli numbering system (Brosius et al., 1981); this region corresponds to positions 2–21 of the 16S rRNA gene sequence (rrnB) of Methanocaldococcus jannaschii (L77117) (Bult et al., 1996). Whole-metagenome sequencing and direct cultivation have shown that some Archaea are not detected when using general Archaea-specific Erlotinib chemical structure primers, including Nanoarchaeum

(Huber et al., 2002) and the ARMAN group (Baker et al., 2006). It is important to assess, redesign and use PCR primers that can amplify more sequences as well as longer sequences for the study of the diversity, distribution and evolution of Archaea. Terrestrial hot springs are an extreme environment where (hyper)thermophilic and/or acidophilic Archaea thrive. The study of the hyperthermophilic Archaea is important to understand the early evolution of life because hyperthermophilic archaeal groups are one of the deepest lineages of all life (Woese, 1987). In fact, the deeper lineage Korarchaeota was detected in a terrestrial hot spring field (Barns et al., 1994; Barns et al., 1996) and the genome analysis has provided an insight into the early evolution of Archaea (Elkins et al., 2008). Several

(hyper)thermophilic Archaea have been cultured from a terrestrial thermoacidic spring in Ohwakudani, Hakone, Japan (Itoh et al., 2002, 2003a, 2007). However, the molecular characterization of this spring field has not Calpain been performed. Here, we report the diversity of archaeal 16S rRNA genes in this spring by PCR-based analysis using a novel Archaea-specific primer modified from Arch21F. Hot water and mud samples were obtained from a thermoacidic spring field that is located in Ohwakudani, Hakone, Japan (35°14.40′N, 139°01.12′E; Supporting Information, Fig. S1), in September 2009. The hot water sample was collected in clean 20-L polypropylene tanks at a hot water pool (78 °C, pH 3.5). The mud sample was collected from a depth of 0–5 cm from the bottom of a warm water pool (28 °C, pH 2.5) that is located downstream of the hot water pool. The mud sample was stored in a sterile 50-mL plastic tube.

, 2003). Ku-0059436 These ‘universal’ primers have been designed based on the conserved regions among most archaeal and/or bacterial 16S rRNA genes. It should be noted that the detectable members are constrained by the nucleotide sequence of the PCR primers used, meaning that these ‘universal’ primers may not be completely universal. Diverse Archaea have been detected in terrestrial and marine environments (Robertson et al., 2005; Schleper et al., 2005). Archaeal diversity in natural environments has often been investigated by PCR-based analysis using Arch21F as a forward primer as reported previously (Delong,

1992) or other primers (Massana et al., 1997; Dojka et al., 1998; Eder et al., 1999; Reysenbach et al., 2000). These primers were designed based on the conserved regions of archaeal 16S rRNA gene sequences between positions 7 and 26 in the Escherichia coli numbering system (Brosius et al., 1981); this region corresponds to positions 2–21 of the 16S rRNA gene sequence (rrnB) of Methanocaldococcus jannaschii (L77117) (Bult et al., 1996). Whole-metagenome sequencing and direct cultivation have shown that some Archaea are not detected when using general Archaea-specific Selleck GSK2126458 primers, including Nanoarchaeum

(Huber et al., 2002) and the ARMAN group (Baker et al., 2006). It is important to assess, redesign and use PCR primers that can amplify more sequences as well as longer sequences for the study of the diversity, distribution and evolution of Archaea. Terrestrial hot springs are an extreme environment where (hyper)thermophilic and/or acidophilic Archaea thrive. The study of the hyperthermophilic Archaea is important to understand the early evolution of life because hyperthermophilic archaeal groups are one of the deepest lineages of all life (Woese, 1987). In fact, the deeper lineage Korarchaeota was detected in a terrestrial hot spring field (Barns et al., 1994; Barns et al., 1996) and the genome analysis has provided an insight into the early evolution of Archaea (Elkins et al., 2008). Several

(hyper)thermophilic Archaea have been cultured from a terrestrial thermoacidic spring in Ohwakudani, Hakone, Japan (Itoh et al., 2002, 2003a, 2007). However, the molecular characterization of this spring field has not Osimertinib chemical structure been performed. Here, we report the diversity of archaeal 16S rRNA genes in this spring by PCR-based analysis using a novel Archaea-specific primer modified from Arch21F. Hot water and mud samples were obtained from a thermoacidic spring field that is located in Ohwakudani, Hakone, Japan (35°14.40′N, 139°01.12′E; Supporting Information, Fig. S1), in September 2009. The hot water sample was collected in clean 20-L polypropylene tanks at a hot water pool (78 °C, pH 3.5). The mud sample was collected from a depth of 0–5 cm from the bottom of a warm water pool (28 °C, pH 2.5) that is located downstream of the hot water pool. The mud sample was stored in a sterile 50-mL plastic tube.

, 2007). However, our microarray hybridization analysis revealed that the mRNA level of the prtA in the mipXcc mutant NK2699 is similar to that in the wild-type strain 8004 (NK2699/8004 = 0.89) (data not shown). This was confirmed by semi-quantitative RT-PCR (Fig. 1a). We also constructed strain mip/pR3PrtA, in which a constitutively expressed prtA was found unable to restore extracellular MK-8669 chemical structure protease activity. It was, however, able to restore activity in 001F10/pR3PrtA (Fig. 1b). The second possibility suggested in our previous article was that MipXcc may be required for the secretion of extracellular proteases (Zang

et al., 2007). Other studies have shown that Xcc’s extracellular enzymes are secreted via the type II secretion system (T2SS) (Hu et al., 1992; Lee et al., 2004). They acquire their native conformations in the periplasmic space before crossing the outer membrane. As shown in Fig. 2a, mature proteases accumulated in the periplasm of the T2SS-deficient mutant strain 258D12. In contrast, no mature

protease was accumulated in the periplasm of the mipXcc mutant. In addition, the prtA mutant did not display any significant protease activity after it was treated with chloroform (Fig. 2a). This indicates that proteases other than PrtA contribute little to the proteolytic activity of Xcc strain 8004. In addition, the portraits of wild-type 8004 and NK2699/pR3MipH6 suggest that not all active protease proteins are secreted Buparlisib ic50 immediately after maturation. Our previous observation that PPIase activity was much less intense in the periplasm of the mipXcc mutant strain than in the wild type suggested that MipXcc might be located in the periplasm of Xcc cells (Zang et al., 2007). In this study, we constructed a complementary strain, NK2699/pR3MipH6, which expressed MipXcc with a 6xHis tag on its C-terminus Sitaxentan (MipH6). As shown in Fig. 2a, the addition of the 6xHis tag to the C-terminus of MipXcc did not affect its function. We prepared the total, periplasmic,

outer membrane and extracellular protein fractions of NK2699/pR3MipH6 during the late log phase. Western blot analysis revealed MipH6 in the total-protein and periplasmic protein fractions but not in the outer membrane or extracellular protein fractions (Fig. 2b). In a parallel experiment, the Zur protein, a transcriptional regulator localized in the cytoplasm of Xcc cells (Huang et al., 2008), was detected only in the total protein fraction but not in the periplasmic and extracellular fractions (Fig. 2b). These results indicate that no cytoplasmic protein was released into the periplasmic or extracellular space. They also demonstrate that MipXcc is located in the periplasm. To determine whether or not MipXcc interacts with PrtA directly, we constructed pTRGMip and pBTPrtA without leader peptides and co-introduced them into BTHrst to create the strain BTHrst/(pBTPrtA-pTRGMip).

, 2007). However, our microarray hybridization analysis revealed that the mRNA level of the prtA in the mipXcc mutant NK2699 is similar to that in the wild-type strain 8004 (NK2699/8004 = 0.89) (data not shown). This was confirmed by semi-quantitative RT-PCR (Fig. 1a). We also constructed strain mip/pR3PrtA, in which a constitutively expressed prtA was found unable to restore extracellular MK-8669 ic50 protease activity. It was, however, able to restore activity in 001F10/pR3PrtA (Fig. 1b). The second possibility suggested in our previous article was that MipXcc may be required for the secretion of extracellular proteases (Zang

et al., 2007). Other studies have shown that Xcc’s extracellular enzymes are secreted via the type II secretion system (T2SS) (Hu et al., 1992; Lee et al., 2004). They acquire their native conformations in the periplasmic space before crossing the outer membrane. As shown in Fig. 2a, mature proteases accumulated in the periplasm of the T2SS-deficient mutant strain 258D12. In contrast, no mature

protease was accumulated in the periplasm of the mipXcc mutant. In addition, the prtA mutant did not display any significant protease activity after it was treated with chloroform (Fig. 2a). This indicates that proteases other than PrtA contribute little to the proteolytic activity of Xcc strain 8004. In addition, the portraits of wild-type 8004 and NK2699/pR3MipH6 suggest that not all active protease proteins are secreted selleck chemicals immediately after maturation. Our previous observation that PPIase activity was much less intense in the periplasm of the mipXcc mutant strain than in the wild type suggested that MipXcc might be located in the periplasm of Xcc cells (Zang et al., 2007). In this study, we constructed a complementary strain, NK2699/pR3MipH6, which expressed MipXcc with a 6xHis tag on its C-terminus Etofibrate (MipH6). As shown in Fig. 2a, the addition of the 6xHis tag to the C-terminus of MipXcc did not affect its function. We prepared the total, periplasmic,

outer membrane and extracellular protein fractions of NK2699/pR3MipH6 during the late log phase. Western blot analysis revealed MipH6 in the total-protein and periplasmic protein fractions but not in the outer membrane or extracellular protein fractions (Fig. 2b). In a parallel experiment, the Zur protein, a transcriptional regulator localized in the cytoplasm of Xcc cells (Huang et al., 2008), was detected only in the total protein fraction but not in the periplasmic and extracellular fractions (Fig. 2b). These results indicate that no cytoplasmic protein was released into the periplasmic or extracellular space. They also demonstrate that MipXcc is located in the periplasm. To determine whether or not MipXcc interacts with PrtA directly, we constructed pTRGMip and pBTPrtA without leader peptides and co-introduced them into BTHrst to create the strain BTHrst/(pBTPrtA-pTRGMip).

35 × 103 CFU per μg DNA when the strain was grown in FOS, and 3.7 × 103 CFU per μg DNA when grown in GOS (Table 2). Plasmid stability was evaluated Etoposide cell line by continuous cultivation for 15 days of five PRL2010 transformants in the

absence of chloramphenicol selection by PCR assays. Notably, all PRL2010 transformants tested did not exhibit any plasmid loss during this period, despite the absence of antibiotic selection. To evaluate the general usefulness of the transformation protocol developed here, we decided to apply it to another Bifidobacterium species, B. asteroides PRL2011, whose genome was recently decoded (F. Bottacini, F. Turroni and M. Ventura, unpublished data). Interestingly, the B. asteroides species represents a distantly related taxon with respect to B. bifidum, while it also occupies a different ecological niche, that is, the hindgut of honeybee (Veerkamp & van Schaik, 1974;

Fischer et al., 1987; Argnani et al., 1996; de Ruyter et al., 1996; Hartke et al., 1996; Rossi et al., 1996; Kullen & Klaenhammer, 2000; Sleator et al., 2001; Schell et al., 2002; Ventura et al., 2006, 2007, 2009; Guglielmetti et al., 2007, 2008; Sela et al., 2008; O’Connell Motherway et al., 2009; Turroni et al., 2010, 2011; Foroni et al., 2011; Serafini et al., 2011). Thus, one may argue that the B. asteroides species possesses a different cell envelope composition (e.g. exopolysaccharides, extracellular proteins) compared to that of B. bifidum. When the transformation protocol optimized on B. bifidum PRL2010 cells was employed for transforming B. asteroides PRL2011 using pNZ8048, a higher transformation efficiency Selleckchem Idasanutlin (1.6 × 104 CFU per μg DNA) was obtained as compared to B. bifidum PRL2010. A direct application from the results of the successful transformation protocol described in this study was to monitor the colonization efficiency of B. bifidum PRL2010 in a murine model. In fact, so far, it has been proven impossible to generate stable antibiotic-resistant B. bifidum PRL2010 derivatives

by spontaneous mutation such as those in other bacterial species might be obtained upon repeated cultivation in the presence of antibiotics. Thus, to discriminate the presence of PRL2010 cells from other members of the gut microbiota of mice, we employed a derivative PRL2010 strain Methocarbamol that contained a plasmid carrying an antibiotic resistance gene to act as a selective marker. The normal microbiota of mice encompasses microorganisms that are sensitive to chloramphenicol (Savino et al., 2011), thus indicating that this antibiotic can be used in selective media. Colonization and clearance of PRL2010 were monitored over a 15-day period by determining viable counts recovered from fecal samples. Two groups of six mice were fed orally on a daily basis with either PRL2010 containing pNZ8048 (designated here as PRL2010pNZ8048) or water for 1 week.

Two recent classical tone-shock conditioning magnetoencephalographic (MEG) studies shed some light on the spatiotemporal characteristics of the so-called conditioned response [CR; a representation of the associated unconditioned stimulus (UCS); Moses et al., 2010] and on the temporal characteristics of shock conditioning and contingency reversal during auditory processing (Kluge et al., 2011). The spatiotemporal dynamics underlying human auditory emotion processing independent of the CR still remain quite elusive. This appears predominantly consequent upon the dynamic

nature of affective sounds revealing their meaning only after signal integration over time (Bradley & Lang, selleck chemical 2000). Bröckelmann et al. (2011) addressed this constraint of signal

convolution by using different ultra-short click-like tones that revealed their identifying characteristic almost instantaneously. Emotional significance was assigned to these tones by means of MultiCS conditioning, a novel and highly challenging affective associative learning procedure (see Steinberg et al., 2012b). Auditory evoked magnetic fields (AEFs) in response to multiple different click-like tones (CS) were compared before and after conditioning with pleasant, unpleasant or neutral auditory scenes (UCS). The results demonstrated the brain’s remarkable capacity to differentiate multiple emotionally relevant from non-relevant tones after brief learning in a rapid and highly resolving fashion. Affect-specific amplified CS processing was evident Z-VAD-FMK clinical trial during the auditory N1m (100–130 ms) and the preceding P20–50 m (20–50 ms) component. Motivated attention, automatically and selectively engaged by emotion-associated tones (Lang et al., 1998a,b; Vuilleumier, 2005), modulated neural activity within a distributed frontal–parietal–temporal

network more generally implicated Sirolimus supplier in the prioritised processing of behaviourally relevant or physically salient stimuli (Corbetta & Shulman, 2002; Fritz et al., 2007). Here, we aimed to investigate whether effects of rapid and highly differentiating affective processing would generalise to cross-modal conditioning of multiple CS with a single electric shock and thus a UCS which is frequently applied in human (Sehlmeyer et al., 2009) and animal neuroscience research. AEFs were measured in response to 40 click-like tones before and after four contingent pairings of 20 stimuli with an electric shock (CS+), while the other half remained unpaired (CS−). Based on our previous findings, we hypothesised a modulation of early AEF components (N1m, P20–50m) within a distributed frontal–parietal–temporal attention network differentiating multiple shock-conditioned tones from unpaired tones. In line with aversive learning studies that reported right-lateralised increased activation to CS+ (Hugdahl et al., 1995; Morris et al., 1997) or greater left-hemispheric responses to CS− (Morris et al., 1998; Rehbein et al.

1). Descriptive baseline characteristics by presence or absence of anal condylomata are shown

in Table 1. The most relevant differences between patients were that a higher percentage of patients with condylomata click here had a history of STIs (46%) and were MSM (84%) compared with patients without condylomata (27% had a history of STIs and 71% were MSM). Accordingly, the percentage of patients practising RAI was also higher in patients with anal condylomata than in those without them (76% vs. 58%, respectively). The overall prevalence of anal condylomata in HIV-infected men was 25% (157 of 640; 95% CI 21–28%). According to sexual behaviour, the prevalence was 28% (132 of 473) in MSM and 15% (25 of 167) in heterosexual HIV-infected men (OR 2.2; 95% CI 1.4–3.5). Condylomatous anal lesions were located in the internal region in 111 of 157 patients (71%), in the perianal area in 13 of 157 patients (8%) Y-27632 solubility dmso and in both locations in 33 of 157 patients (21%) (Fig. 1). The overall prevalence of anal canal HPV infection was 73% (469 of 640; 95% CI 70–77%). The prevalence in patients with anal condylomata was 92% (145 of 157; 95% CI 86–96%) [95.5% (126 of 132) for MSM and 76% (19 of 25) for heterosexuals; χ2 = 11.3; P = 0.001] and that in patients without anal condylomata was 67% (324

of 483; 95% CI 63–71%) [80% (273 of 341) for MSM and 36% (51 of 142) for heterosexuals; χ2 = 88.5; P < 0.001] (with/without anal condylomata, P < 0.001). Moreover,

the prevalence of LR HPV genotypes (63% vs. 19%, respectively; P < 0.001) and that of HR HPV genotypes (83% vs. 62%, respectively; P < 0.001) were considerably higher in the anal canals of HIV-infected men with condylomatous lesions than in those without (Table 2). A higher prevalence of presenting any HPV genotype in the anal canal was associated with having anal condylomata (adjusted OR 8.5; 95% CI 3.2–22). The overall prevalence of single HPV genotype infection was 23% (146 of 640; 95% Fenbendazole CI 20–26%). Similar prevalences of single HPV genotype infection were observed in patients with and without condylomata [18% vs. 24%, respectively; unadjusted OR 0.7; 95% CI 0.4–1.1: in those with condylomata, 14% (19 of 132) for MSM and 36% (nine of 25) for heterosexuals (χ2 = 6.69; P = 0.008); in those without condylomata, 26% (88 of 341) for MSM and 21% (30 of 142) for heterosexuals (χ2 = 1.19; P = 0.275)]. By contrast, the prevalence of HPV infection involving at least two genotypes differed by condylomata status, and this difference was statistically significant: 75% (117 of 157; 95% CI 70–81%) for patients with condylomata [81% (107 of 132) for MSM and 40% (10 of 25) for heterosexuals; χ2 = 18.6; P < 0.001] and 43% (206 of 483; 95% CI 38–47%) for those without condylomata [54% (185 of 341) for MSM and 15% (21 of 142) for heterosexuals; χ2 = 63.8; P < 0.001] (with/without condylomata, adjusted OR 4.0; 95% CI 2.2–7.1).

Vol. 292; pp. 223–224. 2. Saitta D., Antonio F. and Polosa R. Acheieving appropriate regulations for electronic cigarettes. Ther Adv Chronic Dis 2014, 5(2), pp. 50–61. K. Gill, S. Patel Kingston University

London, Surrey, UK The incidence of excessive alcohol consumption is exceptionally high among university students. The study focuses on the potential of alcohol interventions to improve knowledge about CT99021 price sensible drinking. The comparison study found a significant difference of 5.31 between average MCQ results. The United Kingdom was found to consume alcohol excessively compared to its European counterparts. Much of this is contributed by the university student culture where students are known to consume heavily. Recently a new culture of ‘neknomination’ has become popular amongst university students contributing to four deaths. Furthermore previous studies conducted within various UK universities found that the majority of students tested positive for AUDIT-C indicating excessive consumption. Moreover studies have initiated that as little as 5% of students were able to recall the daily

alcohol guidelines.1 Research has indicated that interventions should be initiated, as there have not been many alcohol interventions implemented. The aim of this study was to determine whether a health promotion intervention delivered to university students was effective in improving knowledge about sensible drinking. A comparison study was conducted find more with 100 university students to improve the diversity and precision of results at one university and across two campuses. The students were randomly approached at the reception of both campuses whereby they were screened using an AUDIT-C questionnaire. The final sample consisted of 50 participants from each campus. Then using control conditions, students within the control group were not offered the video based intervention whereas the treatment

arm was. The video was delivered to students via an iPAD, which detailed some facts including the government recommended alcohol guidelines, side effects and the number units in certain alcoholic drinks. In effect an MCQ test consisting of 10 questions that was developed using CPPE packages and piloted with 5 students was given straight after the intervention in the treatment group and as soon as the AUDIT-C questionnaire was completed in the control group Thiamine-diphosphate kinase under untimed conditions. Then 5 students in the treatment group took part in the semi-structured interview due to the limited time availability and student cohort. Those students who agreed to take part were selected randomly via Excel’s random number generator where 5 students were emailed about the interview time and location. Then during the interview 5 open questions were asked where their views and perceptions were recorded about alcohol consumption within students, as well as the effectiveness of the intervention. The faculty’s ethics committee granted ethical approval for this study.

, 2008) (not

shown in Fig. 4 because of the short sequences). The phylotypes in TRG-III were related to environmental clones recovered from acidic wetlands, river water and a mine (Jennifer et al., 2002; Garcia-Moyano et al., 2007; Rowe et al., 2007). TRG-IV includes environmental clones from terrestrial hot springs (Jackson et al., 2001; Ng et al., 2005; Spear et al., 2005). These uncultured phylotypes in the TRGs detected in the present study may represent acidophiles, as supported by the environmental characteristics of the present study field and other environments where related clones were detected, and the physiology of the cultured members of the Thermoplasmata (Reysenbach, 2001). Crenarchaeotic phylotypes

related to cultured thermoacidophiles, such as Thermocladium, Caldisphaera, Metallosphaera, Sulfolobus and Acidianus, were detected in the 28 °C mud sample (Fig. 3). These Lenvatinib mouse cultured thermoacidophiles have been isolated from hot springs (Brock et al., 1972; Segerer et al., 1986; Huber et al., 1989; Itoh et al., 1998, 2003b). These members can grow at a relatively low temperature (45–50 °C) compared with members of Vulcanisaeta, Caldivirga and Stygiolobus (Itoh, 2003), phylotypes of which were detected in hot water samples DAPT and also in the mud sample. Nevertheless, the temperature (28 °C) of the solfataric mud does not provide a suitable growth condition for (hyper)thermophiles. Therefore, these phylotypes related to (hyper)thermophiles that were detected in the mud sample are possibly remnant DNA derived from the high-temperature environments in the hot water pool and/or the stream between the hot water pool and the solfataric mud pool. Phylotypes that did not clearly belong to the cultured thermophilic Crenarchaeota and Euryarchaeota were detected in the mud sample (Fig.

3). These phylotypes were affiliated with the terrestrial hot spring Crenarchaeota (THSC) (Takai & Horikoshi, 1999; Takai & Sako, 1999), Uncultured thermoacidic Spring Clone Group (UTSCG) or Uncultured Thaumarchaeota-related Ribonucleotide reductase clone group (UTRCG). The latter two groups are defined in the present study. These phylotypes were relatively close to the recently proposed Thaumarchaeota (Brochier-Armanet et al., 2008) and Korarchaeota (Barns et al., 1994; Barns et al., 1996) rather than thermophilic cultured Crenarchaeota (Fig. 3). The phylotypes in the THSC (the representative clones are HO28S21A13 and HO28S9A51) were related to environmental clones A14 and A1 (Jackson et al., 2001) and pUWA2 and pUWA36 (Takai & Sako, 1999), which were detected in thermoacidic springs. The phylotype (the representative clone is HO28S9A21) in the UTSCG was related to environmental clones A6 and A13 (Jackson et al., 2001). The phylotypes (the representative clone is HO28S21A56) in the UTRCG were related to soil clone ArcB_cB07 (Hansel et al., 2008) and groundwater clone SWA13 (Shimizu et al., 2007).