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Comparative analysis of hydrogen-producing bacterial biofilms and granular sludge formed in continuous cultures of fermentative bacteria.
Get PDFA system for biohydrogen production was developed based on long-term continuous cultures grown on sugar beet molasses in packed bed reactors. In two separate cultures, consortia of fermentative bacteria developed as biofilms on granitic stones. In one of the cultures, a granular sludge was also formed. Metagenomic analysis of the microbial communities by 454-pyrosequencing of amplified 16S rDNA fragments revealed that the overall biodiversity of the hydrogen-producing cultures was quite small. The stone biofilm from the culture without granular sludge was dominated by Clostridiaceae and heterolactic fermentation bacteria, mainly Leuconostocaeae. Representatives of the Leuconostocaeae and Enterobacteriaceae were dominant in both the granules and the stone biofilm formed in the granular sludge culture. The culture containing granular sludge produced hydrogen significantly more effectively than that containing only the stone biofilm: 5.43 vs. 2.8 mol H2/mol sucrose from molasses, respectively. The speculations that lactic acid bacteria may favor hydrogen production are discussed
Reconstitution of functional mycobacterial arabinosyltransferase AftC proteoliposome and assessment of decaprenylphosphorylarabinose analogues as arabinofuranosyl donors.
Get PDFArabinosyltransferases are a family of membrane-bound glycosyltransferases involved in the biosynthesis of the arabinan segment of two key glycoconjugates, arabinogalactan and lipoarabinomannan, in the mycobacterial cell wall. All arabinosyltransferases identified have been found to be essential for the growth of Mycobcterium tuberculosis and are potential targets for developing new antituberculosis drugs. Technical bottlenecks in designing enzyme assays for screening for inhibitors of these enzymes are (1) the enzymes are membrane proteins and refractory to isolation; and (2) the sole arabinose donor, decaprenylphosphoryl-d-arabinofuranose is sparingly produced and difficult to isolate, and commercial substrates are not available. In this study, we have synthesized several analogues of decaprenylphosphoryl-d-arabinofuranose by varying the chain length and investigated their arabinofuranose (Araf) donating capacity. In parallel, an essential arabinosyltransferase (AftC), an enzyme that introduces α-(1→3) branch points in the internal arabinan domain in both arabinogalactan and lipoarabinomannan synthesis, has been expressed, solubilized, and purified for the first time. More importantly, it has been shown that the AftC is active only when reconstituted in a proteoliposome using mycobacterial phospholipids and has a preference for diacylated phosphatidylinositoldimannoside (Ac(2)PIM(2)), a major cell wall associated glycolipid. α-(1→3) branched arabinans were generated when AftC-liposome complex was used in assays with the (Z,Z)-farnesylphosphoryl d-arabinose and linear α-d-Araf-(1→5)(3-5) oligosaccharide acceptors and not with the acceptor that had a α-(1→3) branch point preintroduced
CK2-mediated phosphorylation of general repressor Maf1 triggers RNA polymerase III activation
Get PDFMaf1 protein is a global negative regulator of Pol III transcription conserved from yeast to man. We report that phosphorylation of Maf1 by casein kinase II (CK2), a highly evolutionarily conserved eukaryotic kinase, is required for efficient Pol III transcription. Both recombinant human and yeast CK2 were able to phosphorylate purified human or yeast Maf1, indicating that Maf1 can be a direct substrate of CK2. Upon transfer of Saccharomyces cerevisiae from repressive to favorable growth conditions, CK2 activity is required for the release of Maf1 from Pol III bound to a tRNA gene and for subsequent activation of tRNA transcription. In a yeast strain lacking Maf1, CK2 inhibition showed no effect on tRNA synthesis, confirming that CK2 activates Pol III via Maf1. Additionally, CK2 was found to associate with tRNA genes, and this association is enhanced in absence of Maf1, especially under repressive conditions. These results corroborate the previously reported TFIIIB-CK2 interaction and indicate an important role of CK2-mediated Maf1 phosphorylation in triggering Pol III activation
Maf1, repressor of RNA polymerase III, indirectly affects tRNA processing
Get PDFMaf1 is negative regulator of RNA polymerase III in yeast. We observed high levels of both primary transcript and end-matured, intron-containing pre-tRNAs in the maf1∆ strain. This pre-tRNA accumulation could be overcome by transcription inhibition, arguing against a direct role of Maf1 in tRNA maturation and suggesting saturation of processing machinery by the increased amounts of primary transcripts. Saturation of the tRNA exportin, Los1, is one reason why end-matured intron-containing pre-tRNAs accumulate in maf1∆ cells. However, it is likely possible that other components of the processing pathway are also limiting when tRNA transcription is increased. According to our model, Maf1- mediated transcription control and nuclear export by Los1 are two major stages of tRNA biosynthesis that are regulated by environmental conditions in a coordinated manner
Purification of recombinant adenovirus type 3 dodecahedric virus-like particles for biomedical applications using short monolithic columns.
Get PDFAdenovirus type 3 dodecahedric virus-like particles (Ad3 VLP) are an interesting delivery vector. They penetrate animal cells in culture very efficiently and up to 300,000 Ad3 VLP can be observed in one cell. The purification of such particles usually consists of several steps. In these work we describe the method development and optimization for the purification of Ad3 VLP using the Convective Interaction Media analytical columns (CIMac). Results obtained with the CIMac were compared to the already established two-step purification protocol for Ad3 VLP based on sucrose density gradient ultracentifugation and the Q-Sepharose ion-exchange column. Pure, concentrated and bioactive VLP were obtained and characterized by several analytical methods. The recovery of the Ad3 VLP was more than 50% and the purified fraction was almost completely depleted of DNA; less than 1% of DNA was present. The purification protocol was shortened from five days to one day and remarkably high penetration efficacy of the CIMac-purified vector was retained. Additionally, CIMac QA analytical column has proven to be applicable for the final and in-process control of various Ad3 VLP sample
Human Suv3 protein reveals its unique features among SF2 helicases
Get PDFSuv3 is a helicase involved in efficient turnover and surveillance of RNA in eukaryotes. In vitro study shows that human Suv3 (hSuv3) in complex with human polynucleotide phosphorylase has RNA degradosome activity. The enzyme is mainly localized in mitochondria; but small fractions are found in cell nuclei. Here we present two X-ray crystallographic structures of human Suv3 in complex with AMPPNP, a non-hydrolysable analog of ATP, and with a short five-nucleotide strand of RNA, at a resolution of 2.08 and 2.9 Å, respectively. The structure of the enzyme is very similar in the two complexes and consists of four domains. Two RecA-like domains form a tandem typical for all helicases from the SF2 superfamily which, together with the C-terminal all-helix domain, makes a ring structure through which the nucleotide strand threads. The N-terminal, mostly helical, domain is positioned externally with respect to the core of the enzyme. Most of the typical helicase motifs are present in hSuv3, but the protein shows certain unique characteristics, suggesting that Suv3 enzymes may constitute a separate subfamily of helicases
Substrate Reduction Therapies for Mucopolysaccharidoses
Get PDFMucopolysaccharidoses (MPS) are inherited metabolic disorders, caused by mutations leading to dysfunction
of one of enzymes involved in degradation of glycosaminoglycans (GAGs) in lysosomes. Due to their impaired degradation, GAGs accumulate in cells of patients, which results in dysfunction of tissues and organs, including the heart, respiratory system, bones, joints and central nervous system. Depending on the kind of deficient enzyme, 11 types and subtypes of MPS are currently recognized. Although enzyme replacement therapy has been developed for 3 types of MPS (types I,II and VI), this treatment was found to be effective only in management of somatic symptoms. Since all MPS types except IVA, IVB and VI are characterized by various problems with functioning of the central nervous system (CNS), a search for effective treatment of this system is highly desirable. Recent discoveries suggested that substrate reduction therapy
may be an efficient method for treatment of MPS patients, including their CNS. In this review, different variants of this therapy will be discussed in the light of recently published reports
Coupling of transcription and replication machineries in the λ DNA replication initiation: evidence for direct interaction of Escherichia coli RNA polymerase and the lambda O protein
Get PDFTranscription proceeding downstream of the lambda phage replication origin was previously shown to support initial steps of the lambda primosome assembly in vitro and to regulate frequency and directionality of lambda DNA replication in vivo. In this report, the data are presented indicating that the RNA polymerase beta subunit makes a direct contact with the lambdaO protein, a replication initiator of lambda phage. These results suggest that the role of RNA polymerase during the initiation of lambda phage DNA replication may be more complex than solely influencing DNA topology. Results demonstrated in this study
also show that gyrase supercoiling activity stimulates the formation of a complex between lambdaO and RNA polymerase, suggesting that the introduction of negative supercoils by DNA gyrase, besides lowering the energy required for DNA strand separation, may play an additional role in modeling
protein–protein interactions at early steps of DNA replication initiation
Yeast Rsp5 ubiquitin ligase affects actin cytoskeleton in vivo and in vitro
Get PDFYeast Rsp5 ubiquitin ligase is involved in several cellular processes, including endocytosis. Actin patches are sites of endocytosis, a process involving actin assembly and disassembly. Here we show Rsp5 localization in cortical patches and demonstrate its involvement in actin cytoskeleton organization and dynamics. We found that Rsp5-F1-GFP2 N-terminal fragment and full length GFP-Rsp5 were recruited to peripheral patches that temporarily co-localized with Abp1-mCherry, a marker of actin patches. Actin cytoskeleton organization was defective in a strain lacking RSP5 or overexpressing RSP5, and this phenotype was accompanied by morphological abnormalities. Overexpression of RSP5 caused hypersensitivity of cells to Latrunculin A, an actin-depolymerizing drug and was toxic to cells lacking Las17, an activator of actin nucleation. Moreover, Rsp5 was required for efficient actin polymerization in a whole cell extract based in vitro system. Rsp5 interacted with Las17 and Las17-binding proteins, Lsb1 and Lsb2, in a GST-Rsp5-WW2/3 pull down assay. Rsp5 ubiquitinated Lsb1-HA and Lsb2-HA without directing them for degradation. Overexpression of RSP5 increased the cellular level of HA-Las17 in wild type and in lsb1Dlsb2D strains in which the basal level of Las17 was already elevated. This increase was prevented in a strain devoid of Las17-binding protein Sla1 which is also a target of Rsp5 ubiquitination. Thus, Rsp5 together with Lsb1, Lsb2 and Sla1 regulate the level of Las17, an important activator of actin polymerization
Nicotiana tabacum EIL2 directly regulates expression of at least one tobacco gene induced by suphur starvation
No full textSulfur deficiency severely affects plant growth and their agricultural productivity leading to diverse changes in development and metabolisms. Molecular mechanisms regulating gene expression under low sulfur conditions remain largely unknown. AtSLIM1, a member of EIN3-like (EIL) family was reported as a central transcriptional regulator of plant sulfur response, however no direct interaction of this protein with any sulfur responsive promoters was demonstrated. The focus of this study was on analysis of a promoter region of UP9C, a tobacco gene strongly induced by sulfur limitation. Cloning and subsequent examination of this promoter resulted in identification of a 20-nt sequence (UPE-box), present also in the promoters of several Arabidopsis genes, including three out of four homologues of UP9C. The UPE-box consists of two parallel tebs sequences (TEIL binding site) proved to be necessary to bind the transcription factors belonging to EIL family and of a 5-nt conserved sequence at the 3’-end. The yeast one-hybrid analysis resulted in identification of one transcription factor capable of binding to the UPE-box - NtEIL2. The interactions of NtEIL2, and its homolog from Arabidopsis, AtSLIM1, with DNA were affected by mutations within the UPE-box. Transient expression assays in Nicotiana benthamiana have further shown that both factors, NtEIL2 and AtSLIM1, activate UP9C promoter. Interestingly, activation by NtEIL2, but not by AtSLIM1, was dependent on sulfur-deficiency of the plants