Likelihood-based significance testing of tree topologies was perf

Likelihood-based significance testing of tree topologies was performed by the pairwise one-sided Kishino–Hasegawa (1sKH) test that has been shown to be superior to the original two-sided Kishino–Hasegawa (2sKH) test (Kishino & Hasegawa, 1989) if evaluated tree topology sets are permutatively incomplete (Goldman et al., 2000) as is the case in the present study. A

set of 297 candidate topologies for significance testing (Table S3) was generated manually in Newick format according to the rationale outlined in Fig. S1. The 1sKH test was performed as implemented in the Tree-Puzzle 5.2 software package applying a 5% significance threshold. Based on the previous phylogenetic analysis of 211 families of single-copy orthologous genes (SCOG) identified in the order Legionellales (Leclerque, 2008a), six genes, namely dnaG, gidA, ksgA, rpoB, rpsA, and sucB (Table S1), were chosen as potential MLST markers as the respective Nutlin-3a SCOG families (i) were found to be sufficiently informative in both phylogenetic analysis and significance testing at the suprageneric level, (ii) at this level clearly fulfilled the dN/dS < 1 criterion, (iii) did not give rise to any detectable sign of lateral gene transfer (LGT) when explored across a set of

alpha- and gammaproteobacterial as well as chlamydial genomes, and (iv) the respective gene loci are widely dispersed across the R. grylli genome. More exactly, all potential protein-encoding MLST markers were located in a single gene copy on the major contig 637 that comprises > 99% of the R. grylli genome sequence (1 581 239 bp). The marker genes are oriented in a way forming three linked marker pairs (ksgA-gidA, rpsA-sucB, dnaG-rpoB), an arrangement that increases the probability to detect

LGT in future studies (Table S2). Moreover, the R. grylli genome contains two identical rRNA operons located at a distance of 370 000 bp from each other on contig 637. Using the primer pairs listed in Table S1, PCR products of expected size (Table S2) were obtained from Rickettsiella pathotypes ‘R. melolonthae’ and ‘R. tipulae’. The triplicate raw sequences generated a reliable consensus for internal partial sequences of genes dnaG, gidA, ksgA, rpoB, rpsA, sucB, and ftsY as well as a 3′-terminal partial sequence of the 23S rRNA-encoding gene rrl. The 16S Amylase rRNA-encoding sequences from both Rickettsiella strains had been determined previously (Leclerque & Kleespies, 2008a, c). Expectedly, amino acid sequences deduced from the protein-encoding marker sequences as well as the rrl nucleotide sequences from both strains unambiguously identified the respective orthologous R. grylli sequence as most closely related GenBank database entry. For each marker, the three Rickettsiella sequences were aligned to two orthologs each from Coxiella and Legionella genomes together with three further gamma- and four alphaproteobacterial as well as three chlamydial orthologs under particular consideration of arthropod-associated bacterial genera.

This might suggest that manipulations of voluntary attention do l

This might suggest that manipulations of voluntary attention do little to speed the process of remapping somatosensory stimuli from anatomical to external spatial coordinates. This possibility is certainly consistent with accounts of somatosensory processing which have characterized the early anatomically based stages of processing as automatic and unconscious (Kitazawa, 2002; Azañón & Soto-Faraco, 2008). In the

study reported here we compared somatosensory processing under conditions in which information about arm posture was provided either by both visual and proprioceptive cues in combination (Exp. 1) or by proprioceptive cues only (Exp. 2). Despite one morphological difference of note – that the P100 and N140 PD-0332991 cost components, which were clearly dissociable in Experiment 1, could not be separately distinguished in Experiment 2 – the SEPs which we observed were largely similar between the two conditions. The effects of posture were observed within 25 ms of one another across the two experiments (Exp. 1 – 128 ms, Exp. 2 – 150 ms). The fact that postural effects can be observed under both of these conditions is consistent with the GSK1120212 chemical structure finding that neurons in primate premotor cortex will remap multisensory correspondences

between touch and vision on the basis of both visual and proprioceptive

cues to posture together and in isolation (e.g. Graziano, 1999). However, the hemispheric distribution of the modulation of the SEPs by posture varied between experiments. Parvulin When participants had sight of their hands as well as signals from proprioception (Exp. 1), an enhancement of the amplitude of the N140 when the hands were across the midline was observed over the contralateral but not the ipsilateral hemisphere. This effect reversed when the participants’ limbs were covered (Exp. 2), with crossed-hands leading to an enhanced N140 recorded over the ipsilateral sites. Because of the differences between the time-windows which we used to compare the N140 across experiments (see above), we examined the Posture × Hemisphere × Experiment interaction with a sample-point by sample-point analysis using a Monte Carlo simulation method (based on Guthrie & Buchwald, 1991). This confirmed that hemispheric variation in posture effects according to the availability of vision of the hand occurred around the N140 component (from 152 ms). This hemispheric variation in posture effects coincides with some prior findings from an fMRI study by Lloyd et al. (2003). Lloyd et al.

coli (38% identity and 50% similarity) and CtpA of B bacilliform

coli (38% identity and 50% similarity) and CtpA of B. bacilliformis (53% identity and 69% similarity) as shown in Fig. GSK1120212 molecular weight 1 (Winsor et al., 2009). An S41 peptidase catalytic domain of 167 residues was recognized in PA5134, characteristic for the S41 peptidase family, as well as a 79-residue PDZ domain upstream of the catalytic domain. PDZ domains are involved in protein–protein interactions and in CTPs interacts with the C-terminus of substrates (Beebe et al., 2000). Serine 302 and lysine 327 were predicted to form the catalytic dyad which corresponds to the S41A subfamily of the MEROPS database (Rawlings et al., 2008). PA3257 was annotated as Prc and showed homology to Prc

of E. coli (44% identity and 60% similarity) and CtpA of B. bacilliformis (32% identity and 50% similarity). In analogy Ibrutinib price to PA5134, Prc has a predicted S41 peptidase catalytic domain of 85 residues downstream of a 175-residue PDZ domain. The MEROPS database classifies PA5134 to the subfamily type S41.004, called C-terminal processing peptidases-3

(CTP-3) and Prc to the subfamily S41.001, called C-terminal processing peptidases-1 (CTP-1) E. coli (Rawlings et al., 2008). A 23-amino acid N-terminal signal peptide was predicted by the signalp program in both CTPs, which indicates a possible translocation across the cytoplasmic membrane by the Sec-pathway (Dyrløv Bendtsen et al., 2004). This prediction is supported by an alkaline phosphatase fusion screen, which identified PA5134 and Prc to cross the inner membrane (Lewenza et al., 2005). The calculated molecular weight of PA5134 without the signal peptide is 43.7 kDa and for 75.6 kDa for Prc in comparison to 44.9 kDa, for CtpA of B. bacilliformis and 74.3 kDa of

E. coli. PA5234 and Prc of P. aeruginosa showed homology with 34% identity and 51% similarity. Interestingly, the genome of P. aeruginosa reveals two CTPs. One, PA5734, showed clear similarity to the CTP-3 subfamily with CtpA of B. bacilliformis as a holotype. The other, PA3257 (Prc), showed similarity to Prc of E. coli belonging to the CTP-1 subfamily. Both predicted Carnitine palmitoyltransferase II proteases contain a catalytic peptidase domain downstream of a PDZ domain although the difference in size between both enzymes is about 31.9 kDa. Figure 1 shows the homology between the CTPs of P. aeruginosa and in comparison with other bacteria. Preliminary blast searches reveal that most Gram-negative bacteria have only one CTP. For example, B. bacilliformis, Legionella pneumophila and Neisseria gonorrhoeae have one CTP protease belonging to the CTP-3 subfamily. CTPs can also be found in the bacteria E. coli, Salmonella enterica and Yersinia pestis. These genomes reveal one CTP belonging to the CTP-1 subfamily. Based on the sequence-predicted protein sizes CTPs of the CTP-3 subfamily constitute the same functional domains but are about 30 kDa smaller than proteases of the CTP-1 subfamily.

The organic

layers were combined and dried using an evapo

The organic

layers were combined and dried using an evaporator at 55 °C. The n-butanol extract was suspended in distilled water, and applied to an MCI GEL CHP20P (75–150 μm) column equilibrated with distilled water. The column was washed extensively with distilled water and then eluted with stepwise gradients of aqueous methanol (30, 50, 70, 90 and 100%, v/v). Each fraction was collected and the antibacterial activity was evaluated using the agar diffusion method (Al-Bayati, 2009). Bacillus subtilis CGMCC 1.1470 was used as the indicator strain. The active fractions eluted with 50% and 70% methanol in water were combined and concentrated. This material was then purified using a preparative HPLC system (Dalian Elite, Dalian, China) equipped with a YMC-pack DOS-A C18 (5 μm, 250 × 20 mm) column. The mobile phase consisted of Milli-Q water containing 0.02% trifluoroacetic I-BET-762 mouse acid and acetonitrile. A triphasic linear gradient of 28–28% acetonitrile (15 min), 28–35% acetonitrile this website (5 min) and 35–45% acetonitrile (40 min) was used for elution at a flow rate of 8 mL min−1. The elution was detected at 210 nm. All isolatable peaks were collected and assessed for antimicrobial activity. The fractions with antimicrobial

activity were vacuum evaporated to dryness. The stability of CFS against heat, pH variation and enzyme treatments was investigated. All experiments were conducted in triplicate. For the heat treatment, CFS was incubated at 40, 60, 80 and 100 °C for 2 h. For pH stability,

CFS was adjusted to pH 1.0–12.0 with HCl or NaOH and held overnight at 4 °C. The treated CFS was neutralized to pH 7.0 before performing an antimicrobial activity assay. To determine its stability against degradative enzymes, CFS was treated with several enzymes at a final concentration of 1 mg mL−1 (Lee et al., 2007). Before the enzymes were added, CFS was adjusted to pH 2.0 for pepsin and pH 7.5 for proteinase K, trypsin and lipase. The reaction mixtures were incubated at 37 °C Carnitine dehydrogenase for 2 h. After the different treatments, the remaining antimicrobial activities of the CFS samples were assessed using the agar diffusion method (Al-Bayati, 2009). Bacillus subtilis CGMCC 1.1470 was used as the indicator strain. The amino acid analyses were carried out using the advanced Marfey’s method with LC/MS. The FDLA derivatives of the purified antibiotics were prepared as described by Fujii et al. (1999). The separation of the l- and d-FDLA derivatives was performed on a ZORBAX SB-C18 (3.5 μm, 150 × 2.1 mm) column with the mobile phase: 20 mM NH4Ac water solution and acetonitrile. A triphasic linear gradient of 10–20% acetonitrile (5 min), 20–50% acetonitrile (35 min) and 50–90% acetonitrile (5 min) was applied at a flow rate of 0.2 mL min−1. The elution pattern was monitored at 340 nm.

1 This research aimed to develop a set of pharmaceutical service

1 This research aimed to develop a set of pharmaceutical service quality indicators that could be further refined into a quality improvement tool for use in both CPs and DDs. A mixed-methods study involving three phases was conducted in south-west England: (1) a survey of CPs and DDs (2) 7 case studies in CP and DD sites using interviews, observation and documentary analysis and (3) a two-round Delphi2 to develop the quality indicators derived from the first two phases. This paper focuses on phase 3. The study received NHS ethical approval. Thematic analysis of phase 2

findings (which had been informed by phase 1) led to the development of 22 quality indicators, which were assessed in a two-round Delphi survey with key stakeholders. Thirty-five key stakeholders were invited to take part, including selleck chemicals llc community pharmacists, dispensing GPs, dispensing assistants/technicians, lay members and board members of CP and DD professional organisations. In round-1, respondents rated the importance for pharmaceutical service quality of Trichostatin A concentration each indicator and suggested possible ways for assessing performance against each indicator. In round-2, respondents were provided with median ratings of importance from round-1 and again rated the importance

of each indicator. Of the 35 people approached 30 (86%) agreed to take part with 22 (63%) completing both rounds. The initial indicators covered communication practices, safety and errors, use of space, training, public health engagement and ethos. In round-1 there was widespread agreement that the indicators captured key areas of service quality and no dimensions were deemed unimportant. For this reason all dimensions were retained in round -2 and an additional indicator, suggested by a participant, was added. Median ratings of the indicators varied little between rounds. There PI-1840 was general agreement of the order of importance

of the four quality themes: safety and dispensing (most important), patient-provider interaction, workplace culture then health promotion. There was disagreement concerning the usefulness of standard operating procedures and the importance of ‘customer service’ issues. Respondents suggested a variety of methods for assessing quality including traditional audits and inspections and more innovative techniques such as mystery shoppers, peer feedback and self-assessment through video playback. A set of 23 quality indicators has been developed for use in CPs and DDs. The indicators highlight certain areas that have received less attention in the past, such as a customer service ethos, as well as re-emphasising the importance of patient safety through safe working practices. Findings suggested a wide variety of ways for assessing service quality, including qualitative and non-traditional methods, which could be used to develop the indicators into a practical resource for practitioners.

In stark contrast to the observation of wild-type cells, examinat

In stark contrast to the observation of wild-type cells, examination of the various mutants indicated that attachment of any of the mutants to any tested surface was almost nonexistent (Fig. 4b shows the result for the flaK mutant on gold grids; others are not shown). In the case of the

flaK mutant (piliated, nonflagellated), a few attached cells were observed compared with the wild type, but only in the case of the nickel grids. In these cases, no cable-like appendages were seen arising from the cells, as expected if these cables are flagella (data not shown). Even after a 48-h incubation, where a large number of wild-type cells had accumulated on silicon, there was still no attachment of any of the mutant cells (Fig. 4c and d for eppA mutant; others not shown). Attachment of wild-type cells appeared to require metabolizing cells, because when the extremely oxygen-sensitive Daporinad ic50 cells were exposed to air for 6 h and then allowed an opportunity to attach to silicon pieces over

3-Methyladenine nmr the course of a further 40-h incubation under aerobic conditions, they did not attach, although both appendages were still observed on the cell surface (data not shown). In addition, a mixture of the flaK mutants with the eppA mutants was also unable to attach to silicon pieces after a 48-h incubation (data not shown). Closer examination of the attached cells demonstrated that they were often tethered to the surfaces by a thick cable of flagella, which often was

observed to unwind to strands of thinner diameter and ultimately to apparently single flagella (Fig. 5). The unwound flagella were most clearly observed when cells were attached to substrates with smooth backgrounds, such as glass and silicon (Fig. 5a and b). Here, one could follow bundles of flagella leaving the cell and then unwinding into thinner bundles and finally to apparently single flagella filaments attached to the substrate. Examination of grids with rougher surfaces, such as nickel, often led to the observation of individual cells attached to the surface in a more three-dimensional setting by multiple flagella cables, while other cables attached ioxilan to neighboring cells (Fig. 5c). Again, the thicker cables could be seen to be unwound to thinner filaments, although this was harder to follow on the rougher surfaces. In some cases, it could be observed that the individual flagella were joining together into the thick bundle as they left the cell (Fig. 6). We attempted to see whether pili production was increased when cells were grown on a surface. As mutants were unable to grow attached to any surface tested, we examined the M. maripaludis flaK mutant after 4-day growth on plates. Cells were scraped off the plates and examined by negative staining. No evidence of increased pili number on the surface of these cells was observed; cells examined typically had only one or two pili and often no pili were observed on cells (data not shown).

Thus, we predict that the role of repeated cocaine exposure would

Thus, we predict that the role of repeated cocaine exposure would have differing effects from the present findings if presented prior to training.

A series of work has now suggested that repeated cocaine exposure prior to learning can result in profound deficits in acquisition. For example, cocaine-treated click here rats have been shown to have impairments in acquiring normal Pavlovian (Schoenbaum & Setlow, 2005; Saddoris et al., 2010) and operant task (Schoenbaum et al., 2004; Calu et al., 2007; Roesch et al., 2007) performance. If animals are unable to learn about cue–outcome or response–outcome associations normally as a result of cocaine exposure (a putatively core-dependent process), then such cocaine exposure should result in impaired, not enhanced, PIT due to poor initial learning, but not because of poor transfer specifically. Given that both the core and shell appear to coordinate activity to produce the PIT effect, it is not known how the core and shell subregions would coordinate activity in the course of learning to produce this phenomenon. Interestingly, many facets of NAc encoding presented here mirror results previously found

PI3K inhibitor in the amygdala. For example, similar to the core, lesions of the basolateral amygdala (BLA) disrupt behavior sensitive to Pavlovian cue encoding in similar tasks (Schoenbaum et al., 1998, 2003b; Balleine et al., 2003; Pickens et al., 2003), while also causing aberrant cue encoding in distally connected regions such as the prefrontal cortex (Schoenbaum et al., 2003a) and NAc (Ambroggi et al., 2008; Jones

et al., 2010). In contrast, the central nucleus of the amygdala (CN) has been shown to be important for attention for learning (Gallagher et al., 1990; Hatfield et al., 1996; Parkinson et al., 2000b; Haney et al., 2010), but less important for detailed cue–outcome associative learning. Consequently, similar to differences between the core and shell in the NAc, BLA and CN show a similar dissociation in PIT. CN lesions abolish potentiating transfer effects, whereas BLA lesions only appear to abolish the behavioral selectivity (i.e. only pressing the CS+-associated lever) of the PIT (Blundell et al., Osimertinib order 2001; Hall et al., 2001; Holland & Gallagher, 2003; Corbit & Balleine, 2005). These core/BLA and shell/CN parallels suggest a larger system by which the amygdala and NAc coordinate activity to produce cue-modulated instrumental behavior. Indeed, BLA inputs to the NAc (Heimer et al., 1991; Brog et al., 1993) appear to be critical for supporting cue-related learning, as asymmetric lesions of the BLA and NAc block the ability for rats to use Pavlovian cues to support new learning (Setlow et al., 2002), whereas inactivation of the BLA selectively alters NAc core encoding during appetitive conditioning (Ambroggi et al.

In contrast to other assays such as the general Streptococcus gen

In contrast to other assays such as the general Streptococcus genus-specific assay targeting the sodA gene, the assay developed check details in this study does not require downstream sequencing for species identification (Poyart et al., 1998). Nevertheless, the primers developed in our study were designed to be compatible with the emerging wide availability of sequencing technologies. Primers 16S-SBSEC-fw and 16S-inf-rev were successfully used in Sanger sequencing performed on two independently obtained amplicons of strain CJ18. RFLP yields the required differentiation power and can be easily performed in-house by most laboratories. However, sequencing can provide an even higher level of detail of the entire

amplicon for subsequent phylogenetic analysis, database comparisons, and potential clustering of isolates. RFLP only differentiates isolates and their amplicons based SB431542 chemical structure on the position of individual restriction enzyme recognition sites but does not deliver information on sequence differences possibly existing between these sites. The RFLP assay performed in separate reactions for MseI and XbaI was consistent among

the reference strains of the SBSEC used in this study. Three RFLP profile groups were distinguished (Fig. 3b): (1) the S. gallolyticus species including Streptococcus alactolyticus featured the expected specific MseI and XbaI profiles; (2) the S. bovis and S. infantarius/S. lutetiensis species were not digested by XbaI and featured the expected group-specific MseI profile; and (3) the S. equinus PCR fragment was digested by XbaI but featured the S. bovis/S. infantarius MseI profile (Table 1 and Fig. 3b). The involvement of members of the SBSEC in food fermentations seems to be larger

than previously expected (Tsakalidou et al., 1998; Díaz-Ruiz et al., 2003; Abdelgadir et al., 2008; Wullschleger, 2009; Jans, 2011). Therefore, the PCR assay developed in this study allows the rapid screening of isolates to identify members of SBSEC within the complex microbial communities of spontaneous food fermentations. Despite a high sequence identity of 98.5% within the amplified DNA fragment, the restriction digestion of PCR products yielded the important discrimination of species into three major SBSEC groups and the differentiation Chlormezanone of the S. gallolyticus cluster (former biotype I and biotype II.2) from the S. bovis/S. infantarius cluster (biotype II.1). This separation is also of clinical relevance because of the association of different infections (Schlegel et al., 2003; Beck et al., 2008). A benefit of the 16S rRNA gene over the groESL is the high conservation and low variability within the 16S rRNA gene that reduces the risk of misidentifying a species, especially when investigating novel and complex microbial niches of previously unstudied sources such as raw dairy products, where diverse microbial communities can be found (Clarridge, 2004; Delbès et al., 2007; Chen et al., 2008; Giannino et al., 2009; Jans, 2011).

Screening of qnr genes was carried out by multiplex PCR amplifica

Screening of qnr genes was carried out by multiplex PCR amplification of qnrA, qnrB and qnrS genes as described (Robicsek et al., 2006). All amplicons obtained

were purified using the Wizard® SV Gel and PCR clean-up system kit (Promega Corporations, Madison, WI). DNA sequencing of purified PCR products was performed by Macrogen (Macrogen Inc., Seoul, Korea). Nucleotide and amino acid sequences were analysed using mega-blast and psi-blast, respectively ( PCR-based Inc/rep typing was performed to identify the major incompatibility groups of the plasmids present in parental and transconjugant strains (Carattoli et al., 2005). Template DNA was prepared by extraction of total DNA using the AG-014699 concentration GenElute™ Bacterial Genomic DNA commercial kit (Sigma). The PCR products obtained were then purified and sequenced as mentioned above.

To identify the relaxase MOB family of the plasmids present in parental and transconjugant strains, a PCR-based MOB amplification method was performed (Alvarado et al., 2008). The primers used to amplify the MOBP13 subfamily were MOBP13 forward (5′-AAC CCA CGC TGC AAR GAY CCV GT-3′) and MOBP13 reverse (5′-AGC GAT GTG selleck compound GAT GTG AAG GTT RTC NGT RTC-3′). PCR conditions were one cycle of denaturation at 94 °C for 4 min, followed by 30 cycles at 94 °C for 30 s, 59 °C for 30 s, 72 °C for 15 s and a final extension at 72 °C for 5 min. The amplified DNA fragments were then purified and sequenced using primers MOBP13 forward and MOBP13 reverse clamp (5′-AGC GAT GTG GAT GTG AAG-3′). From each parental and transconjugant strain, plasmid profiles were visualized after DNA linearization with the S1 enzyme, followed by pulsed-field gel electrophoresis (PFGE) as described previously (Barton et al., 1995). Plasmid sizes were estimated using fingerprinting ii second informatix™ software. S1-PFGE was then transferred onto a nylon-membrane by Southern blotting. Purified DNA products obtained from the PCR of blaDHA-1,

qnrB genes and the replicon IncL/M were used as probes for hybridization of the S1-PFGE blots. These probes were labelled using the commercial kit Amersham ECL Direct Nucleic Acid Labelling and Detection Systems, as recommended by the manufacturer (GE Healthcare). An S. marcescens and an E. coli with an inducible AmpC-β-lactamase phenotype were isolated from a urine sample together with an E. coli with its natural susceptible pattern, a meticillin-resistant Staphylococcus aureus, an Enterococcus faecalis and a Morganella morganii. Primary antibiogram plates of S. marcescens and the resistant E. coli isolate showed oxyimino-β-lactams antagonism with imipenem or cefoxitin. Moreover, we observed scattered colonies located near the edge of cefoxitin, cefotaxime, ceftazidime and aztreonam. This pattern of susceptibility was compatible with the presence of a pACBL (Mirelis et al., 2006).

β-Galactosidase activity due to the core 16S/23S rRNA gene promot

β-Galactosidase activity due to the core 16S/23S rRNA gene promoter in Sulfolobus was 1.7–3-fold lower in the stationary phase than in exponential growth (Fig. 3). The pattern of β-galactosidase activity did not change significantly when normalized for

the absolute copy number of the lacS gene by qPCR, indicating that the increase in activity in exponential growth Ceritinib datasheet was due to regulation of the 16S/23S rRNA gene promoter, not gene dosage (Fig. 3b). The 42-bp 16S/23S rRNA gene core promoter is the smallest reported regulated promoter for Sulfolobus. These findings are consistent with evidence of upregulation of rRNA transcription during exponential growth in E. coli and Saccharomyces cerevisiae (yeast) (Nomura, 1999) and with microarray data from halophilic archaea showing that ribosomal protein gene transcription is higher during exponential growth than in the stationary phase (Lange et al., 2007). Moreover, rRNA in crude preparations from Natronococcus occultus decreases in the stationary phase (Nercessian

& Conde, 2006). The mechanism for core rRNA promoter regulation in S. solfataricus is obscure. The decrease in β-galactosidase activity observed during the stationary phase may be due to growth rate-dependent transcriptional regulation or stringent control in response to decreasing nutrient availability and/or charged tRNAs. The latter has been learn more shown to decrease total stable RNA accumulation in Sulfolobus (Cellini et al., 2004). As in E. coli and yeast, it is likely that there are multiple mechanisms contributing to regulation of the Sulfolobus 16S/23S rRNA gene operon. There is considerable

evidence that archaeal transcriptional regulators interact with core promoters, either binding between or overlapping the TATA box and the transcriptional start site (Peng et al., 2011). In vivo and in vitro analyses have determined several regulatory regions and the start site of the 16S/23S rRNA gene in S. shibatae LY294002 (Hudepohl et al., 1990; Reiter et al., 1990; Hain et al., 1992; Qureshi et al., 1997). The core promoter sequences necessary for transcription initiation in vitro are between −38 and −2 bases relative to the transcription start, identical to those used here in vivo. This region encompasses the proximal promoter element (PPE) (an AT-rich sequence −11 to −2 conserved in Sulfolobus stable RNA promoters), the TATA box, and several bases upstream thereof (Reiter et al., 1990), later identified as a transcription factor B (TFB) recognition element (BRE) (Qureshi & Jackson, 1998). A weak positive regulatory region between −354 and −190 and a negative regulatory region between −93 and −38 were also found (Reiter et al., 1990).