Michael Anbar

Transcriptional Silencing of Multiple Genes in Trophozoites of Entamoeba histolytica

In a previous work we described the transcriptional silencing of the amoebapore A (AP-A) gene (Ehap-a) of Entamoeba histolytica strain HM-1:IMSS. The silencing occurred following transfection with a plasmid containing a 5′ upstream region (473 bp) of Ehap-a that included a truncated segment (140 bp) of a short interspersed nuclear element (SINE1). Silencing remained in effect even after removal of the plasmid (clone G3). Neither short interfering RNA nor methylated DNA were detected, but the chromatin domain of Ehap-a in the gene-silenced trophozoites was modified. Two other similar genes (Ehap-b and one encoding a Saposin-like protein, SAPLIP 1) also became silenced. In the present work we demonstrate the silencing of a second gene of choice, one that encodes the light subunit of the Gal/GalNAc inhibitable lectin (Ehlgl1) and the other, the cysteine proteinase 5 (EhCP-5). This silencing occurred in G3 trophozoites transfected with a plasmid in which the 473 bp 5′ upstream Ehap-a fragment was directly ligated to the second gene. Transcriptional silencing occurred in both the transgene and the chromosomal gene. SINE1 sequences were essential, as was a direct connection between the Ehap-a upstream region and the beginning of the open reading frame of the second gene. Gene silencing did not occur in strain HM-1:IMSS with any of these plasmid constructs. The trophozoites with two silenced genes were virulence-attenuated as were those of clone G3. In addition, trophozoites not expressing Lgl1 and AP-A proteins had a significantly reduced ability to cap the Gal/GalNAc-lectin to the uroid region when incubated with antibodies against the heavy (170 kDa) subunit of the lectin. Lysates of trophozoites lacking cysteine proteinase 5 and AP-A proteins had 30% less cysteine proteinase activity than those of HM-1:IMSS strain or the G3 clone. Silencing of other genes in G3 amoebae could provide a model to study their various functions. In addition, double gene-silenced, virulence-attenuated trophozoites may be an important tool in vaccine development.

Improved Thermostability of Clostridium thermocellum Endoglucanase Cel8A by Using Consensus-Guided Mutagenesis

ABSTRACT The use of thermostable cellulases is advantageous for the breakdown of lignocellulosic biomass toward the commercial production of biofuels. Previously, we have demonstrated the engineering of an enhanced thermostable family 8 cellulosomal endoglucanase (EC 3.2.1.4), Cel8A, from Clostridium thermocellum, using random error-prone PCR and a combination of three beneficial mutations, dominated by an intriguing serine-to-glycine substitution (M. Anbar, R. Lamed, E. A. Bayer, ChemCatChem 2:997–1003, 2010). In the present study, we used a bioinformatics-based approach involving sequence alignment of homologous family 8 glycoside hydrolases to create a library of consensus mutations in which residues of the catalytic module are replaced at specific positions with the most prevalent amino acids in the family. One of the mutants (G283P) displayed a higher thermal stability than the wild-type enzyme. Introducing this mutation into the previously engineered Cel8A triple mutant resulted in an optimized enzyme, increasing the half-life of activity by 14-fold at 85°C. Remarkably, no loss of catalytic activity was observed compared to that of the wild-type endoglucanase. The structural changes were simulated by molecular dynamics analysis, and specific regions were identified that contributed to the observed thermostability. Intriguingly, most of the proteins used for sequence alignment in determining the consensus residues were derived from mesophilic bacteria, with optimal temperatures well below that of C. thermocellum Cel8A.

Enhanced cellulose degradation by targeted integration of a cohesin-fused β-glucosidase into the Clostridium thermocellum cellulosome

The conversion of recalcitrant plant-derived cellulosic biomass into biofuels is dependent on highly efficient cellulase systems that produce near-quantitative levels of soluble saccharides. Similar to other fungal and bacterial cellulase systems, the multienzyme cellulosome system of the anaerobic, cellulolytic bacterium Clostridium thermocellum is strongly inhibited by the major end product cellobiose. Cellobiose-induced inhibition can be relieved via its cleavage to noninhibitory glucose by the addition of exogenous noncellulosomal enzyme β-glucosidase; however, because the cellulosome is adsorbed to the insoluble substrate only a fraction of β-glucosidase would be available to the cellulosome. Towards this end, we designed a chimeric cohesin-fused β-glucosidase (BglA-CohII) that binds directly to the cellulosome through an unoccupied dockerin module of its major scaffoldin subunit. The β-glucosidase activity is thus focused at the immediate site of cellobiose production by the cellulosomal enzymes. BglA-CohII was shown to retain cellobiase activity and was readily incorporated into the native cellulosome complex. Surprisingly, it was found that the native C. thermocellum cellulosome exists as a homooligomer and the high-affinity interaction of BglA-CohII with the scaffoldin moiety appears to dissociate the oligomeric state of the cellulosome. Complexation of the cellulosome and BglA-CohII resulted in higher overall degradation of microcrystalline cellulose and pretreated switchgrass compared to the native cellulosome alone or in combination with wild-type BglA in solution. These results demonstrate the effect of enzyme targeting and its potential for enhanced degradation of cellulosic biomass.

Involvement of a short interspersed element in epigenetic transcriptional silencing of the amoebapore gene in Entamoeba histolytica.

ABSTRACT Transcriptional silencing of an amoebapore (ap-a) gene occurred in Entamoeba histolytica following the transfection of plasmids containing a DNA segment (473 bp) homologous to the 5′ upstream region of the gene (R. Bracha, Y. Nuchamowitz, and D. Mirelman, Eukaryot. Cell 2:295-305, 2003). This segment contains the promoter region of the ap-a gene, a T-rich stretch, followed by a truncated SINE1 (short interspersed element 1) that is transcribed from the antisense strand. Transfection of plasmids containing truncated SINE1 sequences which lack their 3′ regulatory elements upstream of the ap-a gene was essential for the downstream silencing of the ap-a gene while transfection with plasmids containing the entire SINE1 sequence or without the T-rich stretch promoted the overexpression of the ap-a gene. Both the T-rich stretch and sequences of the 5′ SINE1 were essential for the transcription of SINE1. RNA extracts from gene-silenced cultures showed small amounts of short (∼140-nucleotide), single-stranded molecules with homology to SINE1 but no short interfering RNA. Chromatin immunoprecipitation analysis with an antibody against methylated K4 of histone H3 showed a demethylation of K4 at the domain of the ap-a gene, indicating transcriptional inactivation. These results suggest the involvement of SINE1 in triggering the gene silencing and the role of histone modification in its epigenetic maintenance.

Thermostability Enhancement of Clostridium thermocellum Cellulosomal Endoglucanase Cel8A by a Single Glycine Substitution

The thermostability of endoglucanase (E.C. 3.2.1.4) Cel8A, a major component of the cellulosome complex from Clostridium thermocellum, was significantly enhanced using a directed evolution strategy. To ensure that thermostability would not compromise enzyme activity, a two‐step screening strategy was employed that involved consecutive activity and thermostability assays. We have combined three of the mutations from the thermostability screen to obtain a Cel8A variant with a significant increase in thermal resistance without substantial alteration of kinetic parameters. One of the three mutations (S329G) provided the highest contribution to enzyme stability. This single mutation served to increase the Tm by 7.0 °C and the half‐life of activity by eight fold at 85 °C. Site‐saturation mutagenesis at position 329 revealed that only the glycine residue could confer thermostability. The structural changes responsible for the properties of the mutant enzymes are discussed.

Ultrasound Chemical Effects on Pure Organic Liquids.

Molecular fragmentation of organic liquids was produced by cavitation due to ultrasound waves, even in the absence of water. The sonolysis of acetonitrile under argon yielded N2, CH4, and H2; but under oxygen the products were N2, CO, CO2, and H2O. Pure nonaqueous carbon tetrachloride also underwent sonolytic decomposition under either argon or oxygen, with the production of elemental chlorine.

Epigenetic transcriptional gene silencing in Entamoeba histolytica

The human intestinal pathogen Entamoeba histolytica has a number of virulence factors which can cause damage to the host. Transcriptional silencing of the gene coding for one of its major toxic molecules, the amoebapore (Ehap‐a), occurred following the transfection of amoebic trophozoites with a plasmid containing the 5′ promoter region of Ehap‐a as well as a truncated segment of a neighboring, upstream SINE1 element that is transcribed from the opposite strand. Silencing was dependent on the presence of the truncated SINE1 sequences. Small amounts of short (∼140 n), ssRNA molecules with homology to SINE1 were detected in the silenced amoeba but no siRNA. The silenced Ehap‐a gene domain had a chromatin modification indicating transcriptional inactivation without any DNA methylation. Removal of the plasmid did not restore transcription of Ehap‐a. Transcription analysis by microarrays revealed that a number of additional genes were silenced and some were also up‐regulated. Transfections of amoeba which already had a silenced Ehap‐a, with a plasmid containing a second gene ligated to the 5′ upstream region of Ehap‐a, enabled the silencing, in‐trans, of other genes of choice. The nonvirulent phenotype of the gene‐silenced amoeba was demonstrated in various assays and the results suggest that they may have a potential use for vaccination.

Approaches for Improving Thermostability Characteristics in Cellulases

Many efforts have been invested to reduce the cost of biofuel production to substitute renewable sources of energy for fossil-based fuels. At the forefront of these efforts are the initiatives to convert plant-derived cellulosic material to biofuels. Although significant improvements have been achieved recently in cellulase engineering in both efficiency and cost reduction, complete degradation of lignocellulosic material still requires very long periods of time and high enzyme loads. Thermostable cellulases offer many advantages in the bioconversion process, which include increase in specific activity, higher levels of stability, inhibition of microbial growth, increase in mass transfer rate due to lower fluid viscosity, and greater flexibility in the bioprocess. Besides rational design methods, which require deep understanding of protein structure-function relationship, two of the major methods for improvement in specific cellulase properties are directed evolution and knowledge-based library design based on multiple sequence alignments. In this chapter, we provide protocols for constructing and screening of improved thermostable cellulases. Modifications of these protocols may also be used for screening for other improved properties of cellulases such as pH tolerance, high salt, and more.

Hydrogen Isotope Effects in Sonochemical Reactions in Water

Water and dilute (0.1 M) sodium formate solutions of different isotopic composition were subjected to sonolysis under argon in a specially constructed irradiation vessel, using a 0.8 Mc/sec ultrasonic generator. Water containing 50.0 at.% D (pH = 5.5), was subjected to sonolysis and yielded hydrogen with an isotopic composition of 47.0 plus or minus 1% D. No isotope effect within the experimental error could be detected in the molecular hydrogen. DCO2Na (0.1 M), 98% D was sonolyzed in H2O. The hydrogen evolved contained HD/H2 = 0.036 plus or minus 0.002. Formate 0.1 M 50 at.% D yielded under the same conditions HD/H2 0.0103 plus or minus 0.0005. From these values, the isotope effect of hydrogen abstraction was calculated.

Insights into enhanced thermostability of a cellulosomal enzyme

Improved stability of cellulosomal enzymes is of great significance in order to provide efficient degradation of cellulosic derivatives for production of biofuels. In previous reports, we created a quadruple mutant of the endoglucanase Cel8A from Clostridium thermocellum resulting from a combination of both random error-prone PCR and a bioinformatics-based consensus mutagenesis approach. The quadruple mutant exhibited an increased half-life of activity by 14-fold at 85°C with no apparent loss of catalytic activity compared to the wild-type form. Connection of the wild-type enzyme to its respective cohesin partner conferred increased thermostability, but no increase was observed for the cohesin-complexed mutant enzyme. The mutant and the wild-type enzymes were integrated into divalent chimeric scaffoldins with a family 48 exoglucanase partner, and the cellulose-degradation activities of resultant designer cellulosomes were examined. Despite the heightened thermostability of the mutant as a free enzyme, its substitution for the wild-type endoglucanase within the cellulosome context failed to exhibit an improvement in overall degradation of cellulose.