Neither S oralis nor A naeslundii alone were found to form good

Neither S. oralis nor A. naeslundii alone were found to form good biofilms, but growth in the two-species model resulted in abundant mutualistic growth [46]. AI-2 of S. oralis was recently found to be critical for such a mutualistic interaction [6]. Below and above the optimal concentration, mutualistic biofilm growth was suppressed. In S. mutans, LuxS was shown to be PF299 involved in biofilm formation and to affect the structure of biofilms [18, 22, 23], although its role in regulation of factors critical to bacterial adherence and biofilm formation is somewhat controversial. As shown previously, LuxS-deficiency significantly decreased brpA

expression, but no major differences were GSK3326595 price seen between wild-type and the LuxS-deficient mutants in expression of gtfBC, gbpB or spaP [18]. Similar results were also obtained by DNA microarray analysis in both planktonic [47] (Wen et al., unpublished data) and sessile populations (Wen et al., unpublished data). In a study using RealTime-PCR, however, Yoshida et al. [23] reported that transcription of gtfB and gtfC, but not gtfD, was up-regulated

in response to LuxS-deficiency. Like S. mutans and S. oralis, both S. sanguinis http://​www.​oralgen.​lanl.​gov and L. casei (Wen and Burne, unpublished data) possess LuxS. It remains unclear, however, whether LuxS in these bacteria is in fact involved AR-13324 chemical structure in cell-cell communication. Nevertheless, down regulation of luxS expression in S. mutans when grown in dual-species with L. casei and S. oralis would likely affect the absolute

concentration of AI-2 in the biofilms. Studies are ongoing to determine whether AI-2 signaling is functional between these bacterial species and whether alterations in luxS expression does in fact affect the expression of known virulence factors by S. mutans in mixed-species biofilms. It is well established that GtfB and GbpB are critical components of the sucrose-dependent pathway in S. mutans biofilm formation and cariogenicity. In the presence of sucrose, GtfB synthesizes copious Cell press α1,3-linked, water insoluble glucan polymers. Then, surface-associated glucan-binding protein GbpB and others bind to these polymers, facilitating intercellular adherence and biofilm accumulation by S. mutans. It would be expected that down-regulation of GtfB and GbpB would result in less biofilm formation. Surprisingly, our S. mutans-L. casei dual-species data showed that S. mutans accumulated more than 2-fold more biofilms while the expression of gtfB and gbpB was decreased. One possible explanation is that down regulation of GtfB and GbpB (and probably some other members of the Gtfs and Gbps) when grown together with L. casei altered the balance of glucans to glucan-binding proteins ratio or altered the glucan structure in a way that altered biofilm architecture. In fact, similar observations have also been reported recently by us and some other groups [11, 12, 48].

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Phys Rev Lett 2011, 106:220402 CrossRef 6 Fu L, Kane CL: Superco

Phys Rev Lett 2011, 106:220402.CrossRef 6. Fu L, Kane CL: Superconducting proximity effect and check details Majorana fermions at the surface of a topological insulator . Phys Rev Lett 2008, 100:096407.CrossRef 7. Tanaka Y, Yokoyama

T, Nagaosa N: Manipulation of the Majorana fermion, Andreev reflection, and Josephson current on topological insulators . Phys Rev Lett 2009, 103:107002.CrossRef 8. Klinovaja J, Gangadharaiah S, Loss D: Electric-field-induced Majorana Fermions in Armchair Carbon Nanotubes . Phys Rev Lett 2012, 108:196804.CrossRef 9. Read N, Green D: Paired states of fermions in two dimensions with breaking of parity and time-reversal symmetries and the fractional quantum Hall effect . Phys Rev B 2000, 61:10267.CrossRef 10. Potter AC, Lee PA: Majorana end states in multiband microstructures with Rashba spin-orbit coupling . Phys Rev B 2011, 83:094525.CrossRef 11. Wong CLM, Liu J, Law KT, Lee PA: Majorana flat bands and unidirectional Majorana edge states in gapless topological superconductors . Phys Rev B 2013, 88:060504(R).CrossRef 12. Chamon C, Hou C-Y, Mudry C, Ryu S, Santos L: Masses and Majorana fermions

in graphene . Phys. Scr 2012, T146:014013.CrossRef 13. Lutchyn RM, Sau JD, Das SS: Majorana fermions and a topological phase transition in semiconductor-superconductor heterostructures Selleck GDC0449 . Phys Rev Lett 2010, 105:077001.CrossRef 14. Oreg Y, Refael G, von Oppen F: Helical liquids and Majorana bound states

in quantum wires . Phys Rev Lett 2010, 105:177002.CrossRef 15. Mourik V, Zuo K, Frolov SM, Plissard SR, Bakkers EPAM, Kouwenhoven LP: Signatures of Majorana fermions in hybrid superconductorsemiconductor nanowire devices . Science 2012, 336:1003.CrossRef 16. Deng MT, Yu CL, Huang GY, Larsson M, Caroff P, Xu HQ: Anomalous zero-bias conductance peak in a Nb-InSb Nanowire-Nb hybrid device . Nano Lett 2012, 12:6414.CrossRef 17. Das A, Ronen Y, Most Y, Oreg Y, Heiblum M, Shtrikman H: Zero-bias peaks and splitting in an Al-InAs nanowire topological superconductor as a signature of Majorana fermions . Nat Phys 2012, 8:887.CrossRef 18. Lee EJH, Jiang X, Aguado R, Katsaros G, Lieber CM, De FS: Zero-bias anomaly in a nanowire quantum dot coupled to superconductors . Phys Rev Lett 2012, 109:186802.CrossRef 19. Churchill HOH, Fatemi V, Grove-Rasmussen Liothyronine Sodium K, Deng MT, Caroff P, Xu HQ, Marcus CM: Superconductor-nanowire devices from tunneling to the multichannel regime: zero-bias oscillations and magnetoconductance crossover . Phys Rev B 2013, 87:Ricolinostat mw 241401.CrossRef 20. Rokhinson LP, Liu XY, Furdyna JK: The fractional a. c. Josephson effect in a semiconductor-superconductor nanowire as a signature of Majorana particles . Nat Phys 2012, 8:795.CrossRef 21. Law KT, Lee PA, Ng TK: Majorana fermion induced resonant Andreev reflection . Phys Rev Lett 2009, 103:237001.CrossRef 22.

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Current treatments including surgery, chemotherapy, and radiother

Current treatments including surgery, chemotherapy, and radiotherapy remain to have KU55933 clinical trial several disadvantages, thereby often leading to recurrence [2]. Two prophylactic HPV vaccines (Gardasil and Cervarix) [3] can prevent most high-risk HPV infections and minimize the consequences of HPV-associated diseases. However, these vaccines are effective only in adolescents with no history of previous HPV infection and have not shown any therapeutic effects against current HPV infections or associated lesions [3]. Thus, there is an urgent need to develop new specific drugs and methods to treat cervical cancer. Tumor necrosis factor-related

apoptosis-inducing ligand (TRAIL) is a type 2 transmembrane protein that causes apoptosis of target cells through the extrinsic apoptosis pathway. TRAIL selleck products belongs to a member of the tumor necrosis factor superfamily including tumor QNZ order necrosis factor and Fas ligand [4]. The binding of tumor necrosis factor and Fas ligand leads to the damage of normal tissues

in addition to their proapoptotic effect on transformed cells [5, 6], thus limiting their clinical applications. Conversely, TRAIL is able to selectively induce apoptosis in transformed cells but not in most normal cells [4, 7, 8], making it a promising candidate for tumor therapy. Furthermore, tumor growth and progression rely upon angiogenesis [9–11]. Consequently, antiangiogenesis has also emerged as an attractive new strategy in the treatment of cancer [12–16]. Among these agents, endostatin, a 20-kDa C-terminal proteolytic fragment of collagen XVIII, has received the greatest attention Methisazone [17]. It was found not only to be a potent inhibitor of angiogenesis in vitro, but also to have significant antitumor effects in a variety of xenograft-based cancer models and clinical trials [17]. These promising results lead to the rapid advance of this agent into the clinical test [17, 18]. For instance, endostatin enhanced the anticancer effect of CCRT in a mouse xenograft model of cervical cancer [19]. Furthermore, the use of endostatin in combination with other anticancer agents

such as gemcitabine had synergistic antitumor activities [20]. Delivery of therapeutic agents by gene therapy has been extensively studied in a broad range of diseases [21–24]. However, a recurrent problem in these therapies is the efficient delivery of the therapeutic DNA, RNA, or siRNA to the target cells. The key technological impediment to successful gene therapy is vector optimization. Thus, several strategies are being investigated to circumvent this problem such as adeno- or adeno-associated viruses [25]. However, clinical trials have demonstrated substantial obstacles to their use, such as immunogenicity and inflammatory potential [26]. Various non-viral delivery systems, including liposomes [27], dendrimers [28], polycationic polymers [29, 30], and polymeric nanoparticles (NPs) [31] are under development to reduce or avoid immunogenicity and associated risks of toxicity [32].

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Appl Phys Lett 2008, 92:132901–3 CrossRef 30 Liu R: Imaging of p

Appl Phys Lett 2008, 92:132901–3.CrossRef 30. Liu R: Imaging of photoinduced interfacial charge separation in conjugated polymer/semiconductor nanocomposites. J Phys Chem C 2009, 113:9368–9374.CrossRef 31. Diesinger H, Mélin T, Deresmes D, Stiévenard D, Baron T: Hysteretic behavior of the charge injection in single silicon nanoparticles. Appl Phys Lett 2004, 85:3546–3548.CrossRef Competing interests The Protein Tyrosine Kinase inhibitor authors declare that they have no competing interests. Authors’ contributions SW carried out the experiments. ZLW prepared the samples.

SW and XJY interpreted the results and wrote the manuscript. DDL participated in manuscript preparation. ZYZ and ZMJ helped in interpretation and discussions. All authors read and approved the final manuscript.”
“Background Over the past few years, many researchers have shown an interest in silicon GS-9973 molecular weight nanostructures, such as silicon nanocrystals [1–4] and silicon nanowires [5–8] for solar cell applications. Since a silicon nanocrystal embedded in a barrier

material can make carriers confined three-dimensionally, the absorption edge can be tuned in a wide range of photon energies due to the quantum size effect. Thus, it is possible to apply silicon nanocrystal materials or silicon quantum dot (Si-QD) materials Dactolisib nmr to all silicon tandem solar cells [9], which have the possibility to overcome the Shockley-Queisser limit [10]. Moreover, it has Orotidine 5′-phosphate decarboxylase been found that the weak absorption in bulk Si is significantly enhanced in Si nanocrystals, especially in the small dot size, due to the quantum confinement-induced mixing of Γ-character into the X-like conduction band states [11]. Therefore, Si-QD materials are one of the promising materials for the third-generation solar cells. Size-controlled Si-QDs have been prepared in an amorphous silicon oxide (a-SiO2) [12], nitride (a-Si3N4) [13], carbide (a-SiC) [14–17], or hybrid matrix [18, 19], which is called as silicon quantum dot superlattice structure (Si-QDSL). In the case of solar cells, generated carriers have to be transported

to each doping layer. Since the barrier height of an a-SiC matrix is relatively lower than that of an a-Si3N4 or a-SiO2 matrix, the Si-QDSL using an a-SiC matrix has an advantage in carrier transport. Therefore, the development of the Si-QDSL solar cells using an a-SiC matrix is of considerable importance. There are a few researches fabricating Si-QDSL solar cells. Perez-Wurfl et al. reported that Si-QDSL solar cells with SiO2 matrix showed an open-circuit voltage (V oc) of 492 mV. However, the clear evidence of the quantum size effect has not been reported from Si-QDSL solar cells [20]. In our previous work, Si-QDSLs with a-SiC matrix have been prepared by plasma-enhanced chemical vapor deposition (PECVD).

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(D) Nuclear staining of Sox2 in normal

(D) Nuclear staining of Sox2 in normal bronchial epithelium cells, squamous metaplasia and squamous cell carcinomas. (E) Cytoplastic and nuclear staining of Msi2 in normal bronchial epithelium cells, squamous metaplasia and squamous cell

carcinomas. (F) Negative immunostaining signal of Nanog in normal lung, cytoplastic staining of Nanog in squamous metaplasia and squamous cell carcinomas. (G). Negative immunostaining signal of OCT4 in normal lung and tuberculosis, nuclear staining of OCT4 in small cell lung carcinomas. All images were taken at 400× magnification. In non-malignant lung tissues, CD133 was exclusively expressed in some, but not all, bronchial epithelium cells and bronchial selleck screening library smooth muscle cells (Figure 2C). CD133+ bronchial epithelium cells were found in 74% of non-malignant lung tissues while CD133+ bronchial smooth muscle cells were 70%. In lung cancer tissues, about 56% of tumor samples were diffusely positive, 8% focally MK-0518 supplier positive and 2% isolated positive for CD133 (Figure 2C). In non-malignant lung tissues, all bronchial epithelium and squamous metaplasia showed positive expression

of Sox2 (Figure 2D) and Msi2 (Figure 2E), the expression decreases in terminal bronchioles and was absent in alveolar epithelial. In lung cancer, the expression of Sox2 and Msi2 was 90% and 94% respectively, and more than 85% of tissues was diffusely positive for both of the markers (Figure 2D, E). In non-malignant lung tissues, only 2 cases of squamous

metaplasia Selleckchem MK 2206 4-Aminobutyrate aminotransferase in non-tumor adjacent lung tissues were positive for Nanog (Figure 2F), whereas, Nanog staining was detected in 36 of 50 (72%) cases of lung cancer, in which 29 cases were diffusely positive, 6 cases were focally positive and 1 case was isolated positive (Figure 2F). In all non-malignant lung tissues, no positivity for OCT4 was observed (Figure 2G). In lung cancer group, only one case of SCC and one case of SCLC were focally positive for OCT4 (Figure 2G). Potential value of the expression of stem-cell-associated markers as diagnostic markers Table 4 describes the specificity, accuracy and sensitivity of seven stem-cell-associated markers mRNA in bronchoscopic biopsies of lung cancer and non-cancer patients. The stem-cell-associated markers with the highest sensitivity for malignancy were CD44 (98.2%), Sox2 (98.2%) and Msi2 (96.4%), but their specificity were too low to be considered of no clinical significance. Nanog exhibited the highest specificity which was 66.7%, and its sensitivity was 63.4%. Table 4 The specificity, accuracy and sensitivity of seven stem-cell-associated markers mRNA in biopsy samples obtained from bronchoscopy   Specificity, % Accuracy, % Sensitivity, % Bmi1 33.3 80.8 88.4 CD133 44.4 80 85.7 CD44 11.1 86.2 98.2 Sox2 16.7 86.9 98.2 Nanog 66.7 63.8 63.4 OCT4 61.2 82.3 85.7 Msi2 5.6 83.8 96.

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Under glucose abundant conditions (see Figure 1A), the following

Under glucose abundant conditions (see Figure 1A), the following trends can be observed. Both the arcA and iclR knockout strains show an increased selleck chemicals llc biomass yield. When combining these deletions (i.e. in ΔarcAΔiclR) the yield is further increased to 0.63 ± 0.01 c-mole/c-mole glucose, which approximates the theoretical biomass yield of 0.65 c-mole/c-mole glucose (assuming a P/O-ratio of 1.4) [28, 29]. The higher biomass yield is accompanied

by a 70 and 16% reduction in acetate and CO2, respectively. The results of the glucose limited cultures are shown in Figure 1B. The ΔarcAΔiclR strain exhibits an increased biomass yield compared to the wild type strain (0.52 ± 0.01 c-mole/c-mole vs. 0.46 ± 0.01 c-mole/c-mole), but the increment in biomass yield (i.e. 13%) is less distinct

as observed under glucose abundant conditions (47%). The increment in biomass yield is less pronounced under glucose limitation, because glucose limited cultures of the strain ΔarcAΔiclR show a decreased MG-132 datasheet biomass yield while the wild type shows an increased biomass yield compared to if these strains are cultivated under glucose abundant conditions. This can be easily explained: under glucose abundance, the wild type strain converts 16% of the carbon source to acetate as a result of overflow metabolism [30]. At a fixed, low growth rate and consequently under glucose limitation, the cell can easily cope with the delivered carbon and very little carbon is dissipated through formation

of byproducts. However, energy losses also occur in continuous cultures because of the existence of futile cycles [31]. In addition, as shown by Pirt and many others, an excessive fraction of the energy source is reserved for growth-independent maintenance, a factor which is relatively higher under glucose limitation [32–36]. For the wild type cultivated O-methylated flavonoid at a low growth rate (D = ±0.1 h -1), the absence of energy spilling by overflow metabolism compensates and even exceeds the energy spilling by futile cycling and the energy reserved for maintenance, explaining the higher biomass yield observed. In contrast, the ΔarcA ΔiclR strain does not show overflow metabolism under glucose abundance, and therefore the effects of energy loss by futile cycles and maintenance are more visible in this strain leading to a lower biomass yield under glucose limitation. For all experiments in which significantly higher biomass yields were observed, i.e. for ΔiclR in glucose abundant conditions and for ΔarcAΔiclR in glucose abundant and limiting conditions, the high yield is linked to a reduction in CO2 yield.

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Molly K, Woestyne MV, Verstraete W: Development

of a 5-st

Molly K, Woestyne MV, Verstraete W: Development

of a 5-step multichamber reactor as a simulation of the human intestinal microbial ecosystem. Appl Poziotinib mw Microbiol Biotech 1993, 39:254–258.CrossRef 58. Possemiers S, Verthé K, Uyttendaele S, Verstraete W: PCR-DGGE-based quantification of stability of the microbial community in a simulator of the human intestinal microbial ecosystem. FEMS Microbiol Ecol 2004, 49:495–507.PubMedCrossRef 59. van den Abbeele P, Grootaert C, Marzorati M, Possemiers S, Verstraete W, Gérard P, Rabot S, Bruneau A, el Aidy S, Derrien M, Zoetendal E, Kleerebezem M, NU7441 manufacturer Smidt H, van de Wiele T: Microbial community development in a dynamic gut model is reproducible, colon region specific, and selective for Bacteroidetes and Clostridium cluster IX. Appl Environ Microbiol 2010, 76:5237–5246.PubMedCentralPubMedCrossRef 60. van de Wiele T, Boon N, Possemiers S,

Jacobs H, Verstraete W: Prebiotic effects of chicory inulin in the simulator of the human intestinal microbial ecosystem. FEMS Microbiol Ecol 2004, 51:143–153.CrossRef 61. Boon N, Top EM, Verstraete W, Siciliano SD: Bioaugmentation as a tool to protect the structure and function of an activated sludge microbial community against a 3-chloroaniline shock load. Appl Environ Microbiol 2003, 69:1511–1520.PubMedCentralPubMedCrossRef 62. Possemiers S, Bolca S, Grootaert C, Heyerick A, Decroos K, Dhooge W, de Keukeleire D, Rabot S, Verstraete W, van de Wiele

T: The prenylflavonoid isoxanthohumol from hops (Humulus lupulus L.) is activated into the potent phytoestrogen 8-prenylnaringenin in vitro and in the see more very human intestine. J Nutr 2006, 136:1862–1867.PubMed 63. Guo X, Xia X, Tang R, Zhou J, Zhao H, Wang K: Development of a real-time PCR method for Firmicutes and Bacteroidetes in faeces and its application to quantify intestinal population of obese and lean pigs. Lett Appl Microbiol 2008, 47:367–373.PubMedCrossRef 64. Vermeiren J, van den Abbeele P, Laukens D, Vigsnaes LK, de Vos M, Boon N, van de Wiele T: Decreased colonization of fecal Clostridium coccoides/Eubacterium rectale species from ulcerative colitis patients in an in vitro dynamic gut model with mucin environment. FEMS Microbiol Ecol 2012, 79:685–696.PubMedCrossRef 65. Harmsen HJ, Raangs GC, He T, Degener JE, Welling GW: Extensive set of 16S rRNA-based probes for detection of bacteria in human feces. Appl Environ Microbiol 2002, 68:2982–2990.PubMedCentralPubMedCrossRef Competing interests MM, BV, SP, PVdA, WV and TVdW are co-inventor of the pending patent WO2010118857A2. Authors’ contributions MM, VB, SP, PVdA, WV and TVdW developed the concept of the HMI module and designed the experiments; MM performed all the microbiological experiments with the support of MSS and HH for the FISH analyses, of TH for the definition of the permeability of the module and of JP for the computational fluid dynamics simulation.

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: Combined agonist–antagonist genome-wide functional screening id

: Combined agonist–antagonist genome-wide functional screening identifies broadly active antiviral microRNAs. Proc Natl Acad Sci U S A 2010,107(31):13830–13835.PubMedCrossRef 53. Viegas SC, Pfeiffer V, Sittka A, Silva IJ, Vogel J, Arraiano CM: Characterization of the role of ribonucleases in Salmonella small RNA decay. Nucleic Acids Res 2007,35(22):7651–7664.PubMedCrossRef 54. Vogel J, Wagner EG, Gerhart H: Approaches to identify novel non-messenger RNAs in bacteria

and to investigate their biological functions: RNA mining. In Handbook of RNA biochemistry. Edited by: Hartmann RK. Weinheim: Wiley-VCH-Verl; 2005:595–613.CrossRef 55. Datsenko KA, Wanner BL: One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR PLX3397 nmr products. Proc Natl Acad Sci U S A 2000,97(12):6640–6645.PubMedCrossRef Authors’ contributions TS and JY designed CFTRinh-172 all the experiments. JY carried out the experiments. TS and JY wrote the manuscript. Both authors read and approved the final manuscript.”
“Background Heterotrimeric (αβγ) guanine nucleotide binding proteins (G proteins) constitute a family of regulatory GTP hydrolases associated with the cytoplasmic face of the plasma membrane [1–4]. Their activity is characterized

by a cycle of GTP-binding and hydrolysis. The GTP- and GDP-bound complexes define the active and inactive states of the G proteins, respectively. The binding of specific ligands to transmembrane receptors activates the heterotrimeric G protein subunits that are responsible for the flow of BEZ235 price information in many eukaryotic signal transduction pathways

[5]. The traditional G proteins coupled receptors (GPCRs) share a characteristic topological structure of seven transmembrane domains and recognize diverse extracellular signals. The cytoplasmic C-terminal region contains the Gα binding activity. Recently, a new class of seven transmembrane receptors has been identified in humans and other vertebrates and has been classified as belonging to the PAQR superfamily (progestin-adipoQ receptors) [6–10]). Their activity has not been directly associated to heterotrimeric G proteins but indirect Molecular motor evidence suggests that they might be associated to G protein alpha subunits [11, 12]. The PAQR superfamily includes three classes of membrane receptors. Class I PAQRs are adiponectin receptors and include: AdipoR1 (PAQR 1), AdipoR2 (PAQR 2), PAQR 3 and PAQR 6 [13]. These receptors respond to adiponectin that is an insulin-sensitizing peptide hormone found in vertebrates [14, 15]. Low serum adiponectin levels have been identified as a high risk factor for type 2 diabetes and other complications such as atherosclerosis and hepatic steatosis. Adiponectin has been reported to have a positive effect on insulin sensitivity and energy metabolism [16]. Class II PAQRs respond to progesterone and include: mPRα (PAQR 7), mPRβ (PAQR 8) and mPRγ (PAQR 5) [13].

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As described for other fungi [29, 30]

deletion of the P

As described for other fungi [29, 30]

deletion of the P. chrysogenum KU70 homologue increases the frequency of homologous recombination significantly (Marco A. van den Berg, unpublished results). Acetamide-consuming transformants were obtained, purified on fresh media and verified for the correct insertion by PCR. Shake flask experiments demonstrated that the ial null Entinostat datasheet mutant had GSK1904529A order no effect on penicillin production in CP medium supplemented with either precursor, adipate or phenylactetate (103 +/- 1% as compared to both DS17690 and DS54465 strains; 100%). Figure 2 Generation of the ial null mutant in P. chrysogenum. The transcription of the ial gene was blocked by insertion (double crossover; dashed lines) of the amdS selection marker in opposite orientation between the ial gene promoter and the ial ORF. Restriction enzymes indicated: Ba, BamHI; Sb, SbfI; Pm, PmeI. Expression of the ial gene in P. chrysogenum and in vivo role of the IAL in the benzylpenicillin biosynthetic pathway To confirm these results, we carried out different experiments with the engineered strain P. chrysogenum

npe10-AB·C. This strain is a transformed derivative of the npe10 PyrG- strain (Δpen) that contains the pcbAB and pcbC genes, but lacks the wild-type penDE gene [11]. Because of these features, this strain is optimal to assess the putative role of the IAL protein in the benzylpenicillin biosynthetic pathway. The integrity of the ial gene in the learn more npe10-AB·C strain was initially tested by PCR (data not shown) and Southern blotting (Fig. 3A). After digestion of the genomic DNA with HindIII, one 11-kbp band was observed in the npe10-AB·C, size that is coincident with that provided by the Wis54-1255 strain digested with the same MycoClean Mycoplasma Removal Kit restriction enzymes (Fig. 3A). However, after sequencing the ial gene from the npe10-AB·C strain, we found a point mutation at nucleotide 980, where C was changed into T (see Discussion). IPN production by the npe10-AB·C strain was confirmed by HPLC (Fig. 3B). Formation of benzylpenicillin (IPN

acyltransferase activity) and 6-APA (IPN amidohydrolase activity) that might be catalyzed by the IAL, were assessed by growing the npe10-AB·C strain in CP medium. Samples were taken at 48 h and 72 h, but neither 6-APA (Fig. 3C) nor benzylpenicillin (Fig. 3D) were detected by HPLC. This indicates that the npe10-AB·C strain, which contains the ial gene, does not produce these compounds formed in the last step of the penicillin biosynthetic pathway. To test whether the lack of activity is due to a low or null expression rate of the ial gene, northern blot experiments were done with samples taken from the npe10-AB·C and the Wis54-1255 strains grown in CP medium. As shown in Fig. 3E no transcript bands were detected at 24 or 48 h, indicating that this gene is very low or not expressed in P. chrysogenum, in agreement with the absence of detectable ial mRNA in P. chrysogenum NRRL 1951, npe10, Wisconsin54-1255 and DS17690 strains (Marco A.

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Aklujkar, unpublished), form a branch adjacent to succinyl:acetat

Aklujkar, unpublished), form a branch adjacent to succinyl:acetate CoA-transferases of the genus Geobacter (data not shown). In a similar manner, the hypothetical 2-methylcitrate synthase Gmet_1124 and gene Geob_0514 of Geobacter FRC-32 form a branch adjacent

to citrate synthases of Geobacter species (data not shown), consistent with the notion that these two enzyme families could have recently evolved new members capable of converting propionate via propionyl-CoA to 2-methylcitrate. Figure 2 Growth of G. metallireducens on propionate. (a) The gene cluster predicted to encode enzymes of propionate metabolism. (b) The mTOR inhibitor proposed pathway of propionate metabolism. Gmet_0149 (GSU3448) is a homolog of acetate kinase that does not contribute sufficient acetate kinase activity to sustain growth of G. sulfurreducens [17] and has a closer BLAST hit to propionate kinase of E. coli (40% identical sequence) than to acetate kinase of E. coli. Although it does not cluster phylogenetically with either of the E. coli enzymes,

its divergence from acetate kinase (Gmet_1034 = GSU2707) is older than the last common ancestor of the Geobacteraceae (data not shown). This conserved gene product remains to be characterized as a propionate kinase or something else. The proposed pathway for growth of G. metallireducens on propionate (Figure 2) is contingent upon its Selleckchem MLN8237 experimentally established Orotic acid ability to grow on pyruvate [31]. G. sulfurreducens cannot utilize pyruvate as the carbon source unless hydrogen is provided as an electron donor [17]. Oxidation of acetyl-CoA derived from pyruvate in G. sulfurreducens may be prevented by a strict requirement for the succinyl:acetate CoA-transferase reaction (thermodynamically inhibited when acetyl-CoA exceeds acetate) to complete the TCA cycle in the absence of detectable activity of succinyl-CoA synthetase (GSU1058-GSU1059) [17]. With three sets of succinyl-CoA synthetase genes

(Gmet_0729-Gmet_0730, Gmet_2068-Gmet_2069, and Gmet_2260-Gmet_2261), G. metallireducens may produce enough activity to complete the TCA cycle. G. sulfurreducens and G. metallireducens may interconvert malate and pyruvate through a malate oxidoreductase fused to a phosphotransacetylase-like putative regulatory domain (maeB; Gmet_1637 = GSU1700), which is 51% identical to the NADP+-dependent malic enzyme of E. coli [32]. G. sulfurreducens has an additional malate oxidoreductase without this fusion (mleA; GSU2308) that is 53% identical to an NAD+-dependent malic enzyme of B. subtilis [33], but G. metallireducens does not. G. metallireducens possesses orthologous genes for all three pathways that activate pyruvate or oxaloacetate to phosphoenolpyruvate in G. sulfurreducens (Figure 3a): phosphoenolpyruvate synthase (Gmet_0770 = GSU0803), pyruvate phosphate dikinase (Gmet_2940 = GSU0580) and GTP-dependent phosphoenolpyruvate carboxykinase Gmet_2638 = GSU3385) [17].

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