Modeling and Optimization of ß-Galactosidase Entrapping in Polydimethylsiloxane-Modified Silica Composites.

Protein entrapment has multiple applications in enzymatic hydrolysis, drug delivery, etc. Here, we report the studies that successfully utilized the Box-Behnken design to model and optimize the parameters of ß-galactosidase entrapment in sol-gel-derived silica composites. We have also demonstrated the influence of polymer-polydimethylsiloxane as a composite modifying agent on the activity of entrapped enzymes. We have determined how different sol-gel process parameters influence the activity of entrapped enzymes. The highest impact on ß-galactosidase activity was exerted by the water:tetramethoxysilane ratio, followed by polydimethylsiloxane content. Optimized synthesis parameters have been utilized to obtain a composite with maximum ß-galactosidase activity. Performed porosity studies have shown that the addition of polydimethylsiloxane increased the pore diameter. Microscopy studies demonstrated that polydimethylsiloxane-modified composites are softer and less rough. Studies of ß-galactosidase activity using the o-NPG test showed statistically significant shifts in the enzyme temperature and pH profiles compared to the soluble form. An improvement in the reusability of the enzyme and a significant increase in the thermal stability was also observed. When lactose was used, a strong correlation was observed between the substrate concentration and the type of the catalyzed reaction. Moreover, we have demonstrated that the yields and rates of both lactose hydrolysis and galactooligosaccharides formation were correlated with reaction temperature and with the presence of polydimethylsiloxane. All these findings provide the opportunity for industrial use of optimized PDMS-modified silica composites in lactose elimination from dairy products, e.g., milk or whey.

GSH Protects the Escherichia coli Cells from High Concentrations of Thymoquinone.

The aim of the present study was to evaluate the potential protective effect of glutathione (GSH) on Escherichia coli cells grown in a high concentration of thymoquinone (TQ). This quinone, as the main active compound of Nigella sativa seed oil, exhibits a wide range of biological activities. At low concentrations, it acts as an antioxidant, and at high concentrations, an antimicrobial agent. Therefore, any interactions between thymoquinone and glutathione are crucial for cellular defense against oxidative stress. In this study, we found that GSH can conjugate with thymoquinone and its derivatives in vitro, and only fivefold excess of GSH was sufficient to completely deplete TQ and its derivatives. We also carried out studies on cultures of GSH-deficient Escherichia coli strains grown on a minimal medium in the presence of different concentrations of TQ. The strains harboring mutations in gene ΔgshA and ΔgshB were about two- and fourfold more sensitive (256 and 128 µg/mL, respectively) than the wild type. It was also revealed that TQ concentration has an influence on reactive oxygen species (ROS) production in E. coli strains-at the same thymoquinone concentration, the level of ROS was higher in GSH-deficient E. coli strains than in wild type.

Influence of Supercritical Carbon Dioxide Extraction Conditions on Extraction Yield and Composition of Nigella sativa L. Seed Oil-Modelling, Optimization and Extraction Kinetics regarding Fatty Acid and Thymoquinone Content.

Nigella sativa L. is cultivated in many regions and its seeds have found use in variety of foods, but also in traditional medicine due to high content of biologically active essential oils. In this work optimization of supercritical carbon dioxide extraction from N. sativa seeds was performed using response surface methodology to describe the influence of extraction conditions on oil yield. Kinetics of oil and thymoquinone extraction were analyzed as well. It was demonstrated that in order to collect thymoquinone-rich N. sativa oil fraction, appropriate for health-related applications, the extraction should be carried out at 40 °C and 10-15 MPa. Following application of higher pressure of 35 MPa enables effective extraction of remaining oil rich in polyunsaturated fatty acids suitable for use in food industry. Thymoquinone-dependent antibacterial activity of the N. sativa seed oil was observed against bacterial pathogens: Haemophilus influenzae, Staphylococcus haemolyticus, Staphylococcus epidermidis, Enterococcus faecalis and Escherichia coli.

Chromosomal localization of PemIK toxin-antitoxin system results in the loss of toxicity - Characterization of pemIKSa1-Sp from Staphylococcus pseudintermedius.

Toxin-antitoxin (TA) systems are ubiquitous in bacteria and on numerous occasions have been postulated to play a role in virulence of pathogens. Some Staphylococcus aureus strains carry a plasmid, which encodes the highly toxic PemIKSa TA system involved in maintenance of the plasmid but also implicated in modulation of gene expression. Here we showed that pemIKSa1-Sp TA system, homologous to the plasmid-encoded PemIKSa, is present in virtually each chromosome of S. pseudintermedius strain, however exhibits sequence heterogeneity. This results in two length variants of the PemKSa1-Sp toxin. The shorter (96 aa), C-terminally truncated toxin is enzymatically inactive, whereas the full length (112 aa) variant is an RNase, though nontoxic to the host cells. The lack of toxicity of the active PemKSa-Sp2 toxin is explained by increased substrate specificity. The pemISa1-Sp antitoxin gene seems pseudogenized, however, the whole pemIKSa1-Sp system is transcriptionally active. When production of N-terminally truncated antitoxins using alternative start codons is assumed, there are five possible length variants. Here we showed that even substantially truncated antitoxins are able to interact with PemKSa-Sp2 toxin and inhibit its RNase activity. Moreover, the antitoxins can rescue bacterial cells from toxic effects of overexpression of plasmid-encoded PemKSa toxin. Collectively, our data indicates that, contrary to the toxic plasmid-encoded PemIKSa TA system, location of pemIKSa1-Sp in the chromosome of S. pseudintermedius results in the loss of its toxicity. Interestingly, the retained RNase activity of PemKSa1-Sp2 toxin and functionality of the putative, N-terminally truncated antitoxins suggest the existence of evolutionary pressure for alleviation/mitigation of the toxin's toxicity and retention of the inhibitory activity of the antitoxin, respectively.

Double silencing of relevant genes suggests the existence of the direct link between DNA replication/repair and central carbon metabolism in human fibroblasts.

Genetic evidence for a link between DNA replication and glycolysis has been demonstrated a decade ago in Bacillus subtilis, where temperature-sensitive mutations in genes coding for replication proteins could be suppressed by mutations in genes of glycolytic enzymes. Then, a strong influence of dysfunctions of particular enzymes from the central carbon metabolism (CCM) on DNA replication and repair in Escherichia coli was reported. Therefore, we asked if such a link occurs only in bacteria or it is a more general phenomenon. Here, we demonstrate that effects of silencing (provoked by siRNA) of expression of genes coding for proteins involved in DNA replication and repair (primase, DNA polymerase ι, ligase IV, and topoisomerase IIIß) on these processes (less efficient entry into the S phase of the cell cycle and decreased level of DNA synthesis) could be suppressed by silencing of specific genes of enzymes from CMM. Silencing of other pairs of replication/repair and CMM genes resulted in enhancement of the negative effects of lower expression levels of replication/repair genes. We suggest that these results may be proposed as a genetic evidence for the link between DNA replication/repair and CMM in human cells, indicating that it is a common biological phenomenon, occurring from bacteria to humans.

Silencing of the pentose phosphate pathway genes influences DNA replication in human fibroblasts.

Previous reports and our recently published data indicated that some enzymes of glycolysis and the tricarboxylic acid cycle can affect the genome replication process by changing either the efficiency or timing of DNA synthesis in human normal cells. Both these pathways are connected with the pentose phosphate pathway (PPP pathway). The PPP pathway supports cell growth by generating energy and precursors for nucleotides and amino acids. Therefore, we asked if silencing of genes coding for enzymes involved in the pentose phosphate pathway may also affect the control of DNA replication in human fibroblasts. Particular genes coding for PPP pathway enzymes were partially silenced with specific siRNAs. Such cells remained viable. We found that silencing of the H6PD, PRPS1, RPE genes caused less efficient enterance to the S phase and decrease in efficiency of DNA synthesis. On the other hand, in cells treated with siRNA against G6PD, RBKS and TALDO genes, the fraction of cells entering the S phase was increased. However, only in the case of G6PD and TALDO, the ratio of BrdU incorporation to DNA was significantly changed. The presented results together with our previously published studies illustrate the complexity of the influence of genes coding for central carbon metabolism on the control of DNA replication in human fibroblasts, and indicate which of them are especially important in this process.

The dual role of DksA protein in the regulation of Escherichia coli pArgX promoter.

Gene expression regulation by the stringent response effector, ppGpp, is facilitated by DksA protein; however DksA and ppGpp can play independent roles in transcription. In Escherichia coli, the pArgX promoter which initiates the transcription of four tRNA genes was shown to be inhibited by ppGpp. Our studies on the role of DksA in pArgX regulation revealed that it can stimulate transcription by increasing the binding of RNA polymerase to the promoter and the productive transcription complex formation. However, when DksA is present together with ppGpp a severe down-regulation of promoter activity is observed. Our results indicate that DksA facilitates the effects of ppGpp to drive formation of inactive dead-end complexes formed by RNA polymerase at the ArgX promoter. In vivo, ppGpp-mediated regulation of pArgX transcription is dependent on DksA activity. The potential mechanisms of opposing pArgX regulation by ppGpp and DksA are discussed. pArgX is the first reported example of the promoter stimulated by DksA and inhibited by ppGpp in vitro when an overall inhibition occurs in the presence of both regulators. A dual role is thus proposed for DksA in the regulation of the pArgX promoter activity.

Effects of partial silencing of genes coding for enzymes involved in glycolysis and tricarboxylic acid cycle on the enterance of human fibroblasts to the S phase.

BACKGROUND: Previously published reports indicated that some enzymes of the central carbon metabolism (CCM), particularly those involved in glycolysis and the tricarboxylic acid cycle, may contribute to regulation of DNA replication. However, vast majority of such works was performed with the use of cancer cells, in the light of carcinogenesis. On the other hand, recent experiments conducted on bacterial models provided evidence for the direct genetic link between CCM and DNA replication. Therefore, we asked if silencing of genes coding for glycolytic and/or Krebs cycle enzymes may affect the control of DNA replication in normal human fibroblasts. RESULTS: Particular genes coding for these enzymes were partially silenced with specific siRNAs. Such cells remained viable. We found that silencing of certain genes resulted in either less efficient or delayed enterance to the S phase. This concerned following genes: HK2, PFKM, TPI, GAPDH, ENO1, LDHA, CS1, ACO2, SUCLG2, SDHA, FH and MDH2. Decreased levels of expression of HK2, GADPH, CS1, ACO2, FH and MDH2 caused also a substantial impairment in DNA synthesis efficiency. CONCLUSIONS: The presented results illustrate the complexity of the influence of genes coding for enzymes of glycolysis and the tricarboxylic acid cycle on the control of DNA replication in human fibroblasts, and indicate which of them are especially important in this process.

Enzymes of the central carbon metabolism: are they linkers between transcription, DNA replication, and carcinogenesis?

Dependence of carcinogenesis on disruption of DNA replication regulation is a well-known fact. There are also many reports demonstrating the interplay between transcription and DNA replication processes, particularly underlying the importance of promoter activities in the control of replication initiation. Recent findings have shown direct links between central carbon metabolism and DNA replication regulation. Here, we summarize previously published reports which indicated that enzymes of the central carbon metabolism, particularly those involved in glycolysis and the tricarboxylic acid cycle, may contribute to regulation of transcription and DNA transactions (replication and repair). In this light, we propose a hypothesis that some of these enzymes might be linkers between transcription, DNA replication, and carcinogenesis. If true, it may have a consequence in our understanding of causes and mechanisms of carcinogenesis. Particularly, certain metabolic perturbations might directly (through central carbon metabolism enzymes) influence regulation of DNA transactions (replication control and fidelity), and thus facilitate carcinogenesis. To test this hypothesis, further studies will be necessary, which is discussed in the final part of this article.

[Links between glycolysis and DNA replication in eukaryotic cells]. / Powiazanie glikolizy z regulacja replikacji DNA w komórkach eukariotycznych.

Development of the eukaryotic cell proceeds through sequentional stages of the cell cycle, in which there are growth, replication of the genetic material, and cell division processes. Many environmental and intracellular factor decides if the cell proceeds throughout all stages of the cell cycle or enters the G0 silencing phase. The cell cycle depends also on metabolic processes, including central carbon metabolism and DNA replication. One cof major processes of the central carbon metabolism is glycolysis. This is the main pathway of glucose metabolism in cells, leading to conversion of this molecule to puryvare. Until recently, it was supposed that carbon metabolism and DNA replication are linked only indirectly, mostly through energy input, necessary for synthesis of nucleic acids, and production of precursors of deoxyribonucleotides. Nevertheless, recent studies, described and discussed in this article, suggested that there are much more complicated links, perhaps also direct, between these two processes.

A regulatory role for Staphylococcus aureus toxin-antitoxin system PemIKSa.

Toxin-antitoxin systems were shown to be involved in plasmid maintenance when they were initially discovered, but other roles have been demonstrated since. Here we identify and characterize a novel toxin-antitoxin system (pemIKSa) located on Staphylococcus aureus plasmid pCH91. The toxin (PemKSa) is a sequence-specific endoribonuclease recognizing the tetrad sequence U↓AUU, and the antitoxin (PemISa) inhibits toxin activity by physical interaction. Although the toxin-antitoxin system is responsible for stable plasmid maintenance our data suggest the participation of pemIKSa in global regulation of staphylococcal virulence by alteration of the translation of large pools of genes. We propose a common mechanism of reversible activation of toxin-antitoxin systems based on antitoxin transcript resistance to toxin cleavage. Elucidation of this mechanism is particularly interesting because reversible activation is a prerequisite for the proposed general regulatory role of toxin-antitoxin systems.

Characterization of the transcriptional stimulatory properties of the Pseudomonas putida RapA protein.

RNA polymerase-associated factors can significantly affect its performance at specific promoters. Here we identified a Pseudomonas putida RNA polymerases-associated protein as a homolog of Escherichia coli RapA. We found that P. putida RapA stimulates the transcription from promoters dependent on a variety of σ-factors (σ(70), σ(S), σ(54), σ(32), σ(E)) in vitro. The level of stimulation varied from 2- to 10-fold, with the maximal effect observed with the σ(E)-dependent PhtrA promoter. Stimulation by RapA was apparent in the multi-round reactions and was modulated by salt concentration in vitro. However, in contrast to findings with E. coli RapA, P. putida RapA-mediated stimulation of transcription was also evident using linear templates. These properties of P. putida RapA were apparent using either E. coli- or P. putida-derived RNA polymerases. Analysis of individual steps of transcription revealed that P. putida RapA enhances the stability of competitor-resistant open-complexes formed by RNA polymerase at promoters. In vivo, P. putida RapA can complement the inhibitory effect of high salt on growth of an E. coli RapA null strain. However, a P. putida RapA null mutant was not sensitive to high salt. The in vivo effects of lack of RapA were only detectable for the σ(E)-PhtrA promoter where the RapA-deficiency resulted in lower activity. The presented characteristics of P. putida RapA indicate that its functions may extend beyond a role in facilitating RNA polymerase recycling to include a role in transcription initiation efficiency.

Transcription regulation of the Escherichia coli pcnB gene coding for poly(A) polymerase I: roles of ppGpp, DksA and sigma factors.

Poly(A) polymerase I (PAP I), encoded by the pcnB gene, is a major enzyme responsible for RNA polyadenylation in Escherichia coli, a process involved in the global control of gene expression in this bacterium through influencing the rate of transcript degradation. Recent studies have suggested a complicated regulation of pcnB expression, including a complex promoter region, a control at the level of translation initiation and dependence on bacterial growth rate. In this report, studies on transcription regulation of the pcnB gene are described. Results of in vivo and in vitro experiments indicated that (a) there are three σ(70)-dependent (p1, pB, and p2) and two σ(S)-dependent (pS1 and pS2) promoters of the pcnB gene, (b) guanosine tetraphosphate (ppGpp) and DksA directly inhibit transcription from pB, pS1 and pS2, and (c) pB activity is drastically impaired at the stationary phase of growth. These results indicate that regulation of the pcnB gene transcription is a complex process, which involves several factors acting to ensure precise control of PAP I production. Moreover, inhibition of activities of pS1 and pS2 by ppGpp and DksA suggests that regulation of transcription from promoters requiring alternative σ factors by these effectors of the stringent response might occur according to both passive and active models.

ppGpp inhibits the activity of Escherichia coli DnaG primase.

DNA primase is an enzyme required for replication of both chromosomes and vast majority of plasmids. Guanosine tetra- and penta-phosphate (ppGpp and pppGpp, respectively) are alarmones of the bacterial stringent response to starvation and stress conditions, and act by modulation of the RNA polymerase activity. Recent studies indicated that the primase-catalyzed reaction is also inhibited by (p)ppGpp in Bacillus subtilis, where a specific regulation of DNA replication elongation, the replication fork arrest, was discovered. Although in Escherichia coli such a replication regulation was not reported to date, here we show that E. coli DnaG primase is directly inhibited by ppGpp and pppGpp. However, contrary to the B. subtilis primase response to the stringent control alarmones, the E, coli DnaG was inhibited more efficiently by ppGpp than by pppGpp.

Transcription from bacteriophage lambda pR promoter is regulated independently and antagonistically by DksA and ppGpp.

The stringent response effector, guanosine tetraphosphate (ppGpp), adjust gene expression and physiology in bacteria, by affecting the activity of various promoters. RNA polymerase-interacting protein, DksA, was proposed to be the co-factor of ppGpp effects; however, there are reports suggesting independent roles of these regulators. Bacteriophage lambda major lytic promoter, pR, is down-regulated by the stringent response and ppGpp. Here, we present evidence that DksA significantly stimulates pR-initiated transcription in vitro in the reconstituted system. DksA is also indispensable for pR activity in vivo. DksA-mediated activation of pR-initiated transcription is predominant over ppGpp effects in the presence of both regulators in vitro. The possible role of the opposite regulation by ppGpp and DksA in lambda phage development is discussed. The major mechanism of DksA-mediated activation of transcription from pR involves facilitating of RNA polymerase binding to the promoter region, which results in more productive transcription initiation. Thus, our results provide evidence for the first promoter inhibited by ppGpp that can be stimulated by the DksA protein both in vivo and in vitro. Therefore, DksA role could be not only independent but antagonistic to ppGpp in transcription regulation.

IHF- and SeqA-binding sites, present in plasmid cloning vectors, may significantly influence activities of promoters.

Escherichia coli Integration Host Factor (IHF) regulates transcription from some bacterial and phage promoters by affecting DNA topology. Here we demonstrate that IHF affects transcription from bacteriophage lambdapR promoter and the ptac promoter located on plasmids that contain IHF-binding sites. The IHF consensus sites are abundant and they can bind the IHF protein as shown in in vitro studies. The SeqA protein has a role in the complex regulation of pR activity, together with other factors altering DNA topology. Down-regulation of the transcription from ptac in the absence of IHF, together with IHF- and SeqA-mediated effects on pR, suggest that DNA topology cannot be underestimated when assessing in vivo promoters' activity. This may have a significant impact on experiments employing recombinant genes cloned in plasmids and on choosing appropriate plasmid vectors.

Stimulation of the lambda pR promoter by Escherichia coli SeqA protein requires downstream GATC sequences and involves late stages of transcription initiation.

Escherichia coli SeqA protein is a major negative regulator of chromosomal DNA replication acting by sequestration, and thus inactivation, of newly formed oriC regions. However, other activities of this protein have been discovered recently, one of which is regulation of transcription. SeqA has been demonstrated to be a specific transcription factor acting at bacteriophage lambda promoters p(I), p(aQ) and p(R). While SeqA-mediated stimulation of p(I) and p(aQ) occurs by facilitating functions of another transcription activator protein, cII, a mechanism for stimulation of p(R) remains largely unknown. Here, it has been demonstrated that two GATC sequences, located 82 and 105 bp downstream of the p(R) transcription start site, are necessary for this stimulation both in vivo and in vitro. SeqA-mediated activation of p(R) was as effective on a linear DNA template as on a supercoiled one, indicating that alterations in DNA topology are not likely to facilitate the SeqA effect. In vitro transcription analysis demonstrated that the most important regulatory effect of SeqA in p(R) transcription occurs after open complex formation, namely during promoter clearance. SeqA did not influence the appearance and level of abortive transcripts or the pausing during transcription elongation. Interestingly, SeqA is one of few known prokaryotic transcription factors which bind downstream of the regulated promoter and still act as transcription activators.

Bioluminescence-mediated stimulation of photoreactivation in bacteria.

Although biochemistry and genetics of light emission by cells have been investigated in detail, a biological role for bacterial luminescence has remained obscure for a long time. It was proposed recently that luminescence may stimulate DNA repair, but the specific mechanism of this phenomenon was not investigated. Moreover, experiments showing decreased survival of UV-irradiated lux mutants relative to luminescent cells were performed previously using only one bacterial species, Vibrio harveyi. Here, we demonstrate that dark mutants of various strains of naturally luminescent bacteria (Photobacterium leiognathi, Photobacterium phosphoreum and Vibrio fischeri) are more sensitive to UV irradiation than wild-type cells. Thus, this phenomenon occurs not only in V. harveyi but also in other bacterial species. Using an artificial system of luminescent Escherichia coli in combination with phr mutants (defective in photolyase functions), we found that bacterial luminescence may stimulate photoreactivation, perhaps by providing photons that are necessary for photolyase activity.

Sensitivity of dark mutants of various strains of luminescent bacteria to reactive oxygen species.

Recent studies indicated that bioluminescence of the marine bacterium Vibrio harveyi may both stimulate DNA repair and contribute to detoxification of deleterious oxygen derivatives. Therefore, it was also proposed that these reactions can be considered biological roles of bacterial luminescence and might act as evolutionary drives in development of luminous systems. However, experimental evidence for the physiological role of luciferase in protection of cells against oxidative stress has been demonstrated only in one bacterial species, raising the question whether this is a specific or a more general phenomenon. Here we demonstrate that in the presence of various oxidants (hydrogen peroxide, cumene hydroperoxide, t-butyl hydroperoxide and ferrous ions) growth of dark mutants of different strains of Vibrio fischeri and Photobacterium leiognathi is impaired relative to wild-type bacteria, though to various extents. Deleterious effects of oxidants on the mutants could be reduced (with different efficiency) by addition of antioxidants, A-TEMPO or 4OH-TEMPO. These results support the hypotheses that (1) activities of bacterial luciferases may detoxify deleterious oxygen derivatives, and (2) significantly different efficiencies of this reaction are characteristic for various luciferases.