Showing papers in "Journal of Molecular Microbiology and Biotechnology in 2015"
TL;DR: The PTS serves as a complex protein kinase system that regulates a wide variety of transport, metabolic and mutagenic processes as well as the expression of numerous genes in prokaryotes and animals.
Abstract: In 1964, Kundig, Ghosh and Roseman reported the discovery of the phosphoenolpyruvate:sugar phosphotransferase system (PTS), which they subsequently proposed might catalyze sugar transport as well as sugar phosphorylation. What we have learned in the 50 years since its discovery is that, in addition to these primary functions, the PTS serves as a complex protein kinase system that regulates a wide variety of transport, metabolic and mutagenic processes as well as the expression of numerous genes. Recent operon- and genome-sequencing projects have revealed novel PTS protein-encoding genes, many of which have yet to be functionally defined. The current picture of the PTS is that of a complex system with ramifications in all aspects of cellular physiology. Moreover, its mosaic evolutionary history is unusual and intriguing. The PTS can be considered to serve many prokaryotes in capacities of communication and coordination, as do the nervous systems of animals.
63 citations
TL;DR: Evaluating the effect of QS luxS gene on initial biofilm formation by Streptococcus mutans revealed that the wild-type strain tended to form smaller aggregates with uniform distribution, whereas the luxS mutant strain aggregated into distinct clusters easily discernible in the generated biofilm.
Abstract: Quorum sensing (QS) is a process by which bacteria communicate with each other by secreting chemical signals called autoinducers (AIs). Among Gram-negative and Gram-positive bacteria, AI-2 synthesized by the LuxS enzyme is widespread. The aim of this study was to evaluate the effect of QS luxS gene on initial biofilm formation by Streptococcus mutans. The bacterial cell surface properties, including cell hydrophobicity (bacterial adherence to hydrocarbons) and aggregation, which are important for initial adherence during biofilm development, were investigated. The biofilm adhesion assay was evaluated by the MTT method. The structures of the 5-hour biofilms were observed by using confocal laser scanning microscopy, and QS-related gene expressions were investigated by real-time PCR. The luxS mutant strain exhibited higher biofilm adherence and aggregation, but lower hydrophobicity than the wild-type strain. The confocal laser scanning microscopy images revealed that the wild-type strain tended to form smaller aggregates with uniform distribution, whereas the luxS mutant strain aggregated into distinct clusters easily discernible in the generated biofilm. Most of the genes examined were downregulated in the biofilms formed by the luxS mutant strain, except the gtfB gene. QS luxS gene can affect the initial biofilm formation by S. mutans.
38 citations
TL;DR: This study shows that G. resinaceum could be utilized to obtain several bioactive compounds and possess a substantial antiproliferative activity against U937 cells in contrast to G. lucidum, which was shown to have insignificant effects.
Abstract: The aim of this study was to conduct a molecular and biochemical characterization and to compare the antioxidant and antiproliferative activities of four Ganoderma isolates belonging to Ganoderma lucidum (Gl-4, Gl-5) and Ganoderma resinaceum (F-1, F-2) species. The molecular identification was performed by ITS and IGS sequence analyses and the biochemical characterization by enzymatic and proteomic approaches. The antioxidant activity of the ethanolic extracts was compared by three different methods and their flavonoid contents were also analyzed by high-performance liquid chromatography. The antiproliferative effect on U937 cells was determined by MTT assay. The studied mycelia differ both in the enzymatic activities and protein content. The highest content in total phenol and the highest antioxidant activity for DPPH free radical scavenging and chelating activity on Fe(2+) were observed with the Gl-4 isolate of G. lucidum. The presence of quercetin, rutin, myricetin, and morin as major flavonoids with effective antioxidant activity was detected. The ethanolic extracts from mycelia of G. lucidum isolates possess a substantial antiproliferative activity against U937 cells in contrast to G. resinaceum in which the antiproliferative effects were insignificant. This study provides a comparison between G. lucidum and G. resinaceum mycelial strains, and shows that G. resinaceum could be utilized to obtain several bioactive compounds.
26 citations
TL;DR: TP and TS can potentially be used as an alternative to control postharvest fruit diseases caused by R. stolonifer through the induction of H2O2 production, leading to cell membrane oxidative damage and intracellular constituent leakage.
Abstract: The in vitro antifungal activities and mechanism of action of tea polyphenols (TP), tea saponin (TS) and their combination were evaluated against Rhizopus stolonifer . The results sh
25 citations
TL;DR: Amino sugars are dual-purpose compounds in bacteria: they are essential components of the outer wall peptidoglycan and the outer membrane of Gram-negative bacteria and, in addition, when supplied exogenously their catabolism contributes valuable supplies of energy, carbon and nitrogen to the cell.
Abstract: Amino sugars are dual-purpose compounds in bacteria: they are essential components of the outer wall peptidoglycan (PG) and the outer membrane of Gram-negative bacteria and, in addition, when supplied exogenously their catabolism contributes valuable supplies of energy, carbon and nitrogen to the cell. The enzymes for both the synthesis and degradation of glucosamine (GlcN) and N-acetylglucosamine (GlcNAc) are highly conserved but during evolution have become subject to different regulatory regimes. Escherichia coli grows more rapidly using GlcNAc as a carbon source than with GlcN. On the other hand, Bacillus subtilis, but not other Bacilli tested, grows more efficiently on GlcN than GlcNAc. The more rapid growth on this sugar is associated with the presence of a second, GlcN-specific operon, which is unique to this species. A single locus is associated with the genes for catabolism of GlcNAc and GlcN in E. coli, although they enter the cell via different transporters. In E. coli the amino sugar transport and catabolic genes have also been requisitioned as part of the PG recycling process. Although PG recycling likely occurs in B. subtilis, it appears to have different characteristics.
24 citations
TL;DR: Characterization of clostridial phosphotransferases has demonstrated that they are involved in the uptake and phosphorylation of hexoses, hexose derivatives and disaccharides, although the functions of many systems remain to be determined.
Abstract: The acetone-butanol-ethanol fermentation employing solventogenic clostridia was a major industrial process during the 20th century, but declined for economic reasons. In recent times, interest in the process has been revived due to the perceived potential of butanol as a superior biofuel. Redevelopment of an efficient fermentation process will require a detailed understanding of the physiology of carbohydrate utilization by the bacteria. Genome sequences have revealed that, as in other anaerobes, the phosphotransferase system (PTS) and associated regulatory functions are likely to play an important role in sugar uptake and its regulation. The genomes of Clostridium acetobutylicum and C. beijerinckii encode 13 and 43 phosphotransferases, respectively. Characterization of clostridial phosphotransferases has demonstrated that they are involved in the uptake and phosphorylation of hexoses, hexose derivatives and disaccharides, although the functions of many systems remain to be determined. Glucose is a dominant sugar which represses the utilization of other carbon sources, including the non-PTS pentose sugars xylose and arabinose, by the clostridia. Targeting of the CcpA-dependent mechanism of carbon catabolite repression has been shown to be an effective strategy for reducing the repressive effects of glucose, indicating potential for developing strains with improved fermentation performance.
21 citations
TL;DR: The hybrid ANN/GA approach provides a viable alternative to the conventional RSM approach for the modeling and optimization of fermentation processes.
Abstract: Background: Spinosyns, products of secondary metabolic pathway of Saccharopolyspora spinosa, show high insecticidal activity, but difficulty in enhancing the spinosad yield affects wide application. The fermentation process is a key factor in this case. Methods: The response surface methodology (RMS) and artificial neural network (ANN) modeling were applied to optimize medium components for spinosad production using S. spinosa strain CGMCC4.1365. Experiments were performed using a rotatable central composite design, and the data obtained were used to construct an ANN model and an RSM model. Using a genetic algorithm (GA), the input space of the ANN model was optimized to obtain optimal values of medium component concentrations. Results: The regression coefficients (R2) for the ANN and RSM models were 0.9866 and 0.9458, respectively, indicating that the fitness of the ANN model was higher. The maximal spinosad yield (401.26 mg/l) was obtained using ANN/GA-optimized concentrations. Conclusion: The hybrid ANN/GA approach provides a viable alternative to the conventional RSM approach for the modeling and optimization of fermentation processes.
20 citations
TL;DR: How the authors' microbiota influence their feelings, attitudes and mental states with particular reference to autism spectrum disorders is described.
Abstract: Microbial products, released into the bloodstreams of mammals including humans, cross the blood-brain barrier and influence neurodevelopment. They can either promote or alleviate neurological disorders including autism spectrum disorders (ASD). This editorial describes how our microbiota influence our feelings, attitudes and mental states with particular reference to ASD.
20 citations
TL;DR: Physiological-molecular characterization of newly generated unmarked in-frame deletion and complementation mutants allowed defining ChnA (renamed here as Hped) as the enzyme responsible for 1-phenylethanol dehydrogenase, and revealed Hped as a typical homotetrameric SDR.
Abstract: The dehydrogenation of 1-(4-hydroxyphenyl)-ethanol to 4-hydroxyacetophenone represents the second reaction step during anaerobic degradation of p -ethylphenol in the denitrifying bac
20 citations
TL;DR: Main strategies developed to overcome potentially limiting effects in production strains are discussed, including adaptive laboratory evolution selection of PTS- Glc+ mutants, followed by the generation of strains that recover their ability to grow with glucose as a carbon source while allowing the simultaneous consumption of more than one carbon source.
Abstract: Laboratory and industrial cultures of Escherichia coli employ media containing glucose which is mainly transported and phosphorylated by the phosphotransferase system (PTS). In these strains, 50% of the phosphoenolpyruvate (PEP), which results from the catabolism of transported glucose, is used as a phosphate donor for its phosphorylation and translocation by the PTS. This characteristic of the PTS limits the production of industrial biocommodities that have PEP as a precursor. Furthermore, when E. coli is exposed to carbohydrate mixtures, the PTS prevents expression of catabolic and non-PTS transport genes by carbon catabolite repression and inducer exclusion. In this contribution, we discuss the main strategies developed to overcome these potentially limiting effects in production strains. These strategies include adaptive laboratory evolution selection of PTS(-) Glc(+) mutants, followed by the generation of strains that recover their ability to grow with glucose as a carbon source while allowing the simultaneous consumption of more than one carbon source. We discuss the benefits of using alternative glucose transport systems and describe the application of these strategies to E. coli strains with specific genetic modifications in target pathways. These efforts have resulted in significant improvements in the production of diverse biocommodities, including aromatic metabolites, biofuels and organic acids.
19 citations
TL;DR: Some results obtained by the group during recent years on the old phenomenon of diauxie are presented, by means of very fast and quantitative tests, measured in vivo, and obtained from cultures of isogenic mutant strains growing under chemostat conditions, to hint at the problems relating to future PTS research, but also to the ‘true science' of Roseman.
Abstract: Past: The title ‘PTS 50 or The PTS after 50 years' relies on the first description in 1964 of the phosphoenolpyruvate-dependent carbohydrate:phosphotransferase system (PTS) by Kundig, Gosh and Roseman [Proc Natl Acad Sci USA 1964;52:1067-1074]. The system comprised proteins named Enzyme I, HPr and Enzymes II, as part of a novel PTS for carbohydrates in Gram-negative and Gram-positive bacteria, whose ‘biological significance remained unclear'. In contrast, studies which would eventually lead to the discovery of the central role of the PTS in bacterial metabolism had been published since before 1942. They are primarily linked to names like Epps and Gale, J. Monod, Cohn and Horibata, and B. Magasanik, and to phenomena like ‘glucose effects', ‘diauxie', ‘catabolite repression' and carbohydrate transport. Present: The pioneering work from Roseman's group initiated a flood of publications. The extraordinary progress from 1964 to this day in the qualitative and in vitro description of the genes and enzymes of the PTS, and of its multiple roles in global cellular control through ‘inducer exclusion', gene induction and ‘catabolite repression', in cellular growth, in cell differentiation and in chemotaxis, as well as the differences of its functions between Gram-positive and Gram-negative bacteria, was one theme of the meeting and will not be treated in detail here. Future: At the 1988 Paris meeting entitled ‘The PTS after 25 years', Saul Roseman predicted that ‘we must describe these interactions [of the PTS components] in a quantitative way [under] in vivo conditions'. I will present some results obtained by our group during recent years on the old phenomenon of diauxie by means of very fast and quantitative tests, measured in vivo, and obtained from cultures of isogenic mutant strains growing under chemostat conditions. The results begin to hint at the problems relating to future PTS research, but also to the ‘true science' of Roseman.
TL;DR: The results reflect the metabolic significance of rumen-adapted P. ruminicola in utilizing a coarse diet for animals based on acquisition of novel genetic elements.
Abstract: Aim: To reassemble Prevotella ruminicola genome from rumen metagenomic data of cattle and buffalo and compare with the published reference geno
TL;DR: The results suggest that cAMP-dependent catabolite repression, which is mediated by the PTS in E. coli, plays a role in the regulation of biofilm growth in response to sugars.
Abstract: We established that Escherichia coli strain 15 (ATCC 9723) produces both curli and cellulose, and forms robust biofilms. Since this strain is wild type with respect to the phosphoenolpyruvate:sugar phosphotransferase system (PTS), it is an ideal strain in which to investigate the effects of the PTS on the biofilm growth of E. coli. We began by looking into the effects of PTS and non-PTS sugars on the biofilm growth of this strain. All the sugars tested tended to activate biofilm growth at low concentrations but to inhibit biofilm growth at high concentrations. Acidification of the medium was an inhibitory factor in the absence of buffer, but buffering to prevent a pH drop did not prevent the inhibitory effects of the sugars. The concentration at which inhibition set in varied from sugar to sugar. For most sugars, cyclic (c)AMP counteracted the inhibition at the lowest inhibitory concentrations but became ineffective at higher concentrations. Our results suggest that cAMP-dependent catabolite repression, which is mediated by the PTS in E. coli, plays a role in the regulation of biofilm growth in response to sugars. cAMP-independent processes, possibly including Cra, also appear to be involved, in addition to pH effects.
TL;DR: CGL will be a promising enzyme against various cardiovascular diseases and antioxidant deficiency, as well as for generation of a neurotransmitter (H2S) in fungi, with higher affinity for L-cystathionine.
Abstract: Cystathionine γ-lyase (CGL) was purified to its electrophoretic homogeneity from Aspergillus carneus by various chromatographic approaches. The purified enzyme has four identic
TL;DR: This study evaluated the possibility of using GEM (Gram-positive enhancer matrix) particles of Lactobacillus salivarius as a binding platform for 2 conserved, immunodominant, extracytoplasmic Campylobacter jejuni proteins: CjaA and CjaD.
Abstract: Background: Food poisoning and diarrheal diseases continue to pose serious health care and socioeconomic problems worldwide. Campylobacter spp. is a very widespread cause of gastroenteritis. Over the past decade there has been increasing interest in the use of lactic acid bacteria (LAB) as mucosal delivery vehicles. They represent an attractive opportunity for vaccination in addition to vaccination with attenuated bacterial pathogens. Methods: We examined the binding ability of hybrid proteins to nontreated or trichloroacetic acid (TCA)-pretreated LAB cells by immunofluorescence and Western blot analysis. Results: In this study we evaluated the possibility of using GEM (Gram-positive enhancer matrix) particles of Lactobacillus salivarius as a binding platform for 2 conserved, immunodominant, extracytoplasmic Campylobacter jejuni proteins: CjaA and CjaD. We analyzed the binding ability of recombinant proteins that contain C. jejuni antigens (CjaA or CjaD) fused with the protein anchor (PA) of the L. lactis peptidoglycan hydrolase
TL;DR: Growth comparison of D39 ΔgalK with the D39 wild type grown in the presence of galactose shows that galK is required for the proper growth of S. pneumoniae on Galactose.
Abstract: We explored the regulatory mechanism of Leloir pathway genes in Streptococcus pneumoniae D39. Here, we demonstrate that the expression of galKT is galactose dependent. By microarray analysis and quantitative RT-PCR, we further show the role of the transcriptional regulator GalR, present upstream of galKT, as a transcriptional activator of galKT in the presence of galactose. Moreover, we predict a 19-bp regulatory site (5'-GATAGTTTAGTAAAATTTT-3') for the transcriptional regulator GalR in the promoter region of galK, which is also highly conserved in other streptococci. Growth comparison of D39 ΔgalK with the D39 wild type grown in the presence of galactose shows that galK is required for the proper growth of S. pneumoniae on galactose.
TL;DR: The results revealed that P. ramulosum N1 is a promising acidophilic and protease-resistant xylanase-producing microorganism that has great potential to be used in animal feed and food industry applications.
Abstract: Penicillium ramulosum N1 was isolated from decaying wood. This strain produces extracellular xylanases and cellulases. The highest activities of xylanases (250 U/ml) and carboxymethyl cellulose (CMCase; 6.5 U/ml) were produced when 1% barley straw was added as a carbon source. The optimum temperature and pH for xylanase activity was 55 and 3.0 °C, respectively. The xylanases exhibited strong protease resistance. CMCase revealed maximum activities at pH 3.0 and in the range of 60-70 °C. Filter paper activity was optimally active at pH 5.0 and 55 °C. The zymograms produced by the SDS-PAGE resolution of the crude enzymes indicated that there are four bands of protein with xylanase activity and three bands of proteins with endoglucanase. The results revealed that P. ramulosum N1 is a promising acidophilic and protease-resistant xylanase-producing microorganism that has great potential to be used in animal feed and food industry applications.
TL;DR: The results suggest that the chicken fecal excreted microbiome is a dynamic system with a differentiated population structure that harbors a highly restricted number of higher taxa.
Abstract: Poultry contamination can be largely attributed to the presence of chicken feces during the production process. Fecal contamination is often found in raw chicken products sold for human consumption. Quantitative analysis of the fecal microbial community of chickens using next-generation sequencing techniques is the focus of this study. Fecal samples were collected from 30 broiler chickens at two time points: days 1 and 35 of development. 454 pyrosequencing was conducted on 16S rRNA extracted from each sample, and microbial population dynamics were investigated using various automated bioinformatics pipelines. Diversity of the microbial community at the genus level increased during the 5-week growth period. Despite this growth, only a few dominant bacteria groups (over 80%) were identified in each fecal sample, with most groups being unique and only a few were shared between samples. Population analysis at the genus level showed that microbial diversity increased with chicken growth and development. Classification and phylogenetic analysis of highly represented microbes (over 1%) clearly showed high levels of sequence similarity between groups such as Firmicutes and Proteobacteria. These results suggest that the chicken fecal excreted microbiome is a dynamic system with a differentiated population structure that harbors a highly restricted number of higher taxa.
TL;DR: The hexitol D-mannitol is transported by many bacteria via a phosphoenolpyruvate (PEP):carbohydrate phosphotransferase system (PTS) and, despite strong sequence conservation, the mechanisms controlling the activity of MtlR from Lactobacillus casei, Bacillus subtilis and Geobacillin stearothermophilus are quite different.
Abstract: The hexitol D-mannitol is transported by many bacteria via a phosphoenolpyruvate (PEP):carbohydrate phosphotransferase system (PTS). In most Firmicutes, the transcription activator MtlR controls the expression of the genes encoding the D-mannitol-specific PTS components and D-mannitol-1-P dehydrogenase. MtlR contains an N-terminal helix-turn-helix motif followed by an Mga-like domain, two PTS regulation domains (PRDs), an EIIB(Gat)- and an EIIA(Mtl)-like domain. The four regulatory domains are the target of phosphorylation by PTS components. Despite strong sequence conservation, the mechanisms controlling the activity of MtlR from Lactobacillus casei, Bacillus subtilis and Geobacillus stearothermophilus are quite different. Owing to the presence of a tyrosine in place of the second conserved histidine (His) in PRD2, L. casei MtlR is not phosphorylated by Enzyme I (EI) and HPr. When the corresponding His in PRD2 of MtlR from B. subtilis and G. stearothermophilus was replaced with alanine, the transcription regulator was no longer phosphorylated and remained inactive. Surprisingly, L. casei MtlR functions without phosphorylation in PRD2 because in a ptsI (EI) mutant MtlR is constitutively active. EI inactivation prevents not only phosphorylation of HPr, but also of the PTS(Mtl) components, which inactivate MtlR by phosphorylating its EIIB(Gat)- or EIIA(Mtl)-like domain. This explains the constitutive phenotype of the ptsI mutant. The absence of EIIB(Mtl)-mediated phosphorylation leads to induction of the L. caseimtl operon. This mechanism resembles mtlARFD induction in G. stearothermophilus, but differs from EIIA(Mtl)-mediated induction in B. subtilis. In contrast to B. subtilis MtlR, L. casei MtlR activation does not require sequestration to the membrane via the unphosphorylated EIIB(Mtl) domain.
TL;DR: An effective means of nitrilase immobilization via spore surface display is demonstrated, which can be applied in biological processes or conversion and showed a higher tolerance for adverse environmental conditions.
Abstract: Nitrilases are an important class of industrial enzymes. They require mild reaction conditions and are highly efficient and environmentally friendly, so they are used to catalyze the synthesis of carboxylic acid from nitrile, a process considered superior to conventional chemical syntheses. Nitrilases should be immobilized to overcome difficulties in recovery after the reaction and to stabilize the free enzyme. The nitrilase from Clostridium thermocellum was expressed, identified and displayed on the surface of Bacillus subtilis spores by using the spore coat protein G of B. subtilis as an anchoring motif. In a free state, the recombinant nitrilase catalyzed the conversion of 3-cyanopyridine to niacin and displayed maximum catalytic activity (8.22 units/mg protein) at 40 °C and pH 7.4. SDS-PAGE and Western blot were used to confirm nitrilase display. Compared with the free enzyme, the spore-immobilized nitrilase showed a higher tolerance for adverse environmental conditions. After the reaction, recombinant spores were recovered via centrifugation and reused 3 times to catalyze the conversion of 3-cyanopyridine with 75.3% nitrilase activity. This study demonstrates an effective means of nitrilase immobilization via spore surface display, which can be applied in biological processes or conversion.
TL;DR: Results suggest that overexpression of the aminotransferase-encoding NCgl0462 open reading frame plays an unequivocal role in the L-ornithine biosynthetic pathway, with overlapping substrate specificity in C. glutamicum.
Abstract: Overexpression of the NCgl0462 open reading frame, encoding a class II aminotransferase, was studied in conjunction with other enzymes in L-ornithine biosynthesis in an L-ornithine-producing strain. Expression of the wild-type NCgl0462 open reading frame, which displayed aminotransferase activity, was amplified by placing it under the control of the glyceraldehyde 3-phosphate dehydrogenase gene promoter in the pEK0 plasmid and in the genome. L-Ornithine production in Corynebacteriumglutamicum SJC8260 harboring plasmid and the genomic NCgl0462 open reading frame was increased by 8.8 and 21.6%, respectively. In addition, the combined overexpression of the NCgl0462 open reading frame within the genome along with the mutated L-ornithine biosynthesis genes (argCJBD) placed in the pEK0 plasmid in C. glutamicum SJC8260 resulted in significant improvement in L-ornithine production (12.48 g/l for combined overexpression compared with 8.42 g/l for the control). These results suggest that overexpression of the aminotransferase-encoding NCgl0462 open reading frame plays an unequivocal role in the L-ornithine biosynthetic pathway, with overlapping substrate specificity in C. glutamicum.
TL;DR: The data suggest that this cross-talk becomes most relevant under conditions that would inhibit activity of EINtr and accordingly modulate processes controlled by EIIA.
Abstract: Many Proteobacteria possess the regulatory nitrogen-related phosphotransferase system (PTS(Ntr)), which operates in parallel to the transport PTS. PTS(Ntr) is composed of the proteins EI(Ntr) and NPr and the final phosphate acceptor EIIA(Ntr). Both PTSs can exchange phosphoryl groups among each other. Proteins governing K(+) uptake represent a major target of PTS(Ntr) in Escherichia coli. Nonphosphorylated EIIA(Ntr) binds and stimulates the K(+) sensor KdpD, which activates expression of the kdpFABC operon encoding a K(+) transporter. Here we show that this regulation also operates in an ilvG(+) strain ruling out previous concern about interference with a nonfunctional ilvG allele present in many strains. Furthermore, we analyzed phosphorylation of EIIA(Ntr). In wild-type cells EIIA(Ntr) is predominantly phosphorylated, regardless of the growth stage and the utilized carbon source. However, cross-phosphorylation of EIIA(Ntr) by the transport PTS becomes apparent in the absence of EI(Ntr): EIIA(Ntr) is predominantly nonphosphorylated when cells grow on a PTS sugar and phosphorylated when a non-PTS carbohydrate is utilized. These differences in phosphorylation are transduced into corresponding kdpFABC transcription levels. Thus, the transport PTS may affect phosphorylation of EIIA(Ntr) and accordingly modulate processes controlled by EIIA(Ntr). Our data suggest that this cross-talk becomes most relevant under conditions that would inhibit activity of EI(Ntr).
TL;DR: Overall, IVET was demonstrated as a tool for the identification of in vivo-induced promoter elements of B. burgdorferi, and the observed infection bottleneck apparent using a polyclonal infection pool provides insight into the dynamics of experimental infection with the causative agent of Lyme disease.
Abstract: The causative agent of Lyme disease, Borrelia burgdorferi, is an obligate parasite that requires either a tick vector or a mammalian host for survival. Identification of the bacterial genes that are specifically expressed during infection of the mammalian host could provide targets for novel therapeutics and vaccines. In vivo expression technology (IVET) is a reporter-based promoter trap system that utilizes selectable markers to identify promoters of bacterial host-specific genes. Using previously characterized genes for in vivo and in vitro selection, this study utilized an IVET system that allows for selection of B. burgdorferi sequences that act as active promoters only during murine infection. This promoter trap system was able to successfully distinguish active promoter sequences both in vivo and in vitro from control sequences and a library of cloned B. burgdorferi genomic fragments. However, a bottleneck effect during the experimental mouse infection limited the utility for genome-wide promoter screening. Overall, IVET was demonstrated as a tool for the identification of in vivo-induced promoter elements of B. burgdorferi, and the observed infection bottleneck apparent using a polyclonal infection pool provides insight into the dynamics of experimental infection with B. burgdorferi.
TL;DR: The role of the transcriptional regulator FcsR, present upstream of the fcs operon, is demonstrated as a transcriptional activator of the FscR regulatory site and the expression of various genes and operons was altered in the presence of fucose.
Abstract: In this study, we explore the impact of fucose on the transcriptome of S. pneumoniae D39. The expression of various genes and operons, including the fucose uptake PTS and utilization operon (fcs operon) was altered in the presence of fucose. By means of quantitative RT-PCR and β-galactosidase analysis, we demonstrate the role of the transcriptional regulator FcsR, present upstream of the fcs operon, as a transcriptional activator of the fcs operon. We also predict a 19-bp putative FcsR regulatory site (5'-ATTTGAACATTATTCAAGT-3') in the promoter region of the fcs operon. The functionality of this predicted FcsR regulatory site was further confirmed by promoter-truncation experiments, where deletion of half of the FscR regulatory site or full deletion led to the abolition of expression of the fcs operon.
TL;DR: A low-cost and uncomplicated method for the production and purification of biologically active hbFGF fusion proteins that have the ability to support several culture passages of undifferentiated human embryonic stem cells and induce pluripotent stem cells.
Abstract: To reduce the cost of cytokines and growth factors in stem cell research, a simple method for the production of soluble and biological active human basic fibroblast growth factor (hbFGF) fusion protein in Escherichia coli was established. Under optimal conditions, approximately 60-80 mg of >95% pure hbFGF fusion proteins (Trx-6xHis-hbFGF and 6xHis-hbFGF) were obtained from 1 liter of culture broth. The purified hbFGF proteins, both with and without the fusion tags, were biologically active, which was confirmed by their ability to stimulate proliferation of NIH3T3 cells. The fusion proteins also have the ability to support several culture passages of undifferentiated human embryonic stem cells and induce pluripotent stem cells. This paper describes a low-cost and uncomplicated method for the production and purification of biologically active hbFGF fusion proteins.
TL;DR: Results are presented supporting the novel suggestion that in certain instances the phosphoenolpyruvate:sugar phosphotransferase system also regulates the mutation rate, which involves the regulation of insertion sequence-5 hopping into a specific site upstream of the glpFK glycerol utilization operon in Escherichia coli.
Abstract: The phosphoenolpyruvate:sugar phosphotransferase system (PTS) has been shown to control transport, cell metabolism and gene expression. We here present results supporting the novel suggestion that in certain instances it also regulates the mutation rate. Directed mutations are defined as mutations that occur at higher frequencies when beneficial than when neutral or detrimental. To date, the occurrence of directed point mutations has not been documented and confirmed, but a few examples of transposon-mediated directed mutations have been reported. Here we focus on the first and best-studied example of directed mutation, which involves the regulation of insertion sequence-5 hopping into a specific site upstream of the glpFK glycerol utilization operon in Escherichia coli. This insertional event specifically activates expression of the glpFK operon, allowing the growth of wild-type cells with glycerol as a carbon source in the presence of nonmetabolizable glucose analogues which normally block glycerol utilization. The sugar-transporting PTS controls this process by regulating levels of cytoplasmic glycerol-3-phosphate and cyclic (c)AMP as established in previous publications. Direct involvement of the glycerol repressor, GlpR, and the cAMP receptor protein, Crp, in the regulation of transposon-mediated directed mutation has been demonstrated.
TL;DR: A three-dimensional model of angiotensin-converting enzyme from Stigmatella aurantiaca is built and it is suggested that catalytic residue Glu 434 of the model could play a crucial role to degrade Aβ peptide between Asp 7 and Ser 8 and could be used to design new therapeutic strategies against Alzheimer's disease.
Abstract: Amyloid-β (Aβ)-degrading enzymes are known to degrade Aβ peptides, a causative agent of Alzheimer's disease. These enzymes are responsible for maintaining Aβ concentration. However, loss of such enzymes or their Aβ-degrading activity because of certain genetic as well as nongenetic reasons initiates the accumulation of Aβ peptides in the human brain. Considering the limitations of the human enzymes in clearing Aβ peptide, the search for microbial enzymes that could cleave Aβ is necessary. Hence, we built a three-dimensional model of angiotensin-converting enzyme (ACE) from Stigmatella aurantiaca using homology modeling technique. Molecular docking and molecular dynamics simulation techniques were used to outline the possible cleavage mechanism of Aβ peptide. These findings suggest that catalytic residue Glu 434 of the model could play a crucial role to degrade Aβ peptide between Asp 7 and Ser 8. Thus, ACE from S. aurantiaca might cleave Aβ peptides similar to human ACE and could be used to design new therapeutic strategies against Alzheimer's disease.
TL;DR: The lipase showed tolerance to various solvents as well as activity at low temperature, therefore, this lipase might be of great potential to be employed in various industrial applications.
Abstract: Background: Bacillus lipases are grouped in subfamily 1.4, which are the smallest known lipases having a molecular mass of 19.6 kDa. Lipases act
TL;DR: The demonstration of a CPO isogene which is expressed as a secreted and active CPO clarifies the nature of this isoenzyme already identified in earlier reports.
Abstract: Inspection of transcriptome data from the chloroperoxidase (CPO)-producing fungus Caldariomyces fumago DSM1256 led to the discovery of two distinct CPO mRNA sequences. This strain could be shown to contain the newly identified isogene as well as produce and secrete both isoenzymes. The CPO2 enzyme bears high sequence similarity to the well-characterized CPO (87% identity for the mature proteins). It shows two insertions in the signal peptide and in the C-terminal propeptide, and one deletion in the mature polypeptide close to the C-terminus. Furthermore, it lacks one of the serine residues known to be O-glycosylated in the CPO sequence. The demonstration of a CPO isogene which is expressed as a secreted and active CPO clarifies the nature of this isoenzyme already identified in earlier reports. A structure model comparison shows a high conservation of the active site and the substrate channel, suggesting very similar catalytic properties.
TL;DR: Data is presented on the construction of an inducible system for expression of the lytic peptidases AlpA and AlpB from Lysobacter sp.
Abstract: Development of an efficient expression system for (especially secreted) bacterial lytic enzymes is a complicated task due to the specificity of their action. The substrate for such enzymes is peptidoglycan, the main structural component of bacterial cell walls. For this reason, expression of recombinant lytic proteins is often accompanied with lysis of the producing bacterium. This paper presents data on the construction of an inducible system for expression of the lytic peptidases AlpA and AlpB from Lysobacter sp. XL1 in Pseudomonas fluorescens Q2-87, which provides for the successful secretion of these proteins into the culture liquid. In this system, the endopeptidase gene under control of the T7lac promoter was integrated into the bacterial chromosome, as well as the Escherichia coli lactose operon repressor protein gene. The T7 pol gene under lac promoter control, which encodes the phage T7 RNA polymerase, is maintained in Pseudomonas cells on the plasmids. Media and cultivation conditions for the recombinant strains were selected to enable the production of AlpA and AlpB by a simple purification protocol. Production of recombinant lytic enzymes should contribute to the development of new-generation antimicrobial drugs whose application will not be accompanied by selection of resistant microorganisms.