Expression in Pichia pastoris of Thermostable Endo-1,4-ß-xylanase from the Actinobacterium Nocardiopsis halotolerans: Properties and Use for Saccharification of Xylan-Containing Products.

A gene encoding a polysaccharide-degrading enzyme was cloned from the genome of the bacterium Nocardiopsis halotolerans. Analysis of the amino acid sequence of the protein showed the presence of the catalytic domain of the endo-1,4-ß-xylanases of the GH11 family. The gene was amplified by PCR and ligated into the pPic9m vector. A recombinant producer based on Pichia pastoria was obtained. The production of the enzyme, which we called NhX1, was carried out in a 10 L fermenter. Enzyme production was 10.4 g/L with an activity of 927 U/mL. Purification of NhX1 was carried out using Ni-NTA affinity chromatography. The purified enzyme catalyzed the hydrolysis of xylan but not other polysaccharides. Endo-1,4-ß-xylanase NhX1 showed maximum activity and stability at pH 6.0-7.0. The enzyme showed high thermal stability, remaining active at 90 °C for 20 min. With beechwood xylan, the enzyme showed Km 2.16 mg/mL and Vmax 96.3 U/mg. The products of xylan hydrolysis under the action of NhX1 were xylobiose, xylotriose, xylopentaose, and xylohexaose. Endo-1,4-ß-xylanase NhX1 effectively saccharified xylan-containing products used for the production of animal feed. The xylanase described herein is a thermostable enzyme with biotechnological potential produced in large quantities by P. pastoria.

Untranslated Region Sequences and the Efficacy of mRNA Vaccines against Tuberculosis.

mRNA vaccines have been shown to be effective in combating the COVID-19 pandemic. The amount of research on the use of mRNAs as preventive and therapeutic modalities has undergone explosive growth in the last few years. Nonetheless, the issue of the stability of mRNA molecules and their translation efficiency remains incompletely resolved. These characteristics of mRNA directly affect the expression level of a desired protein. Regulatory elements of RNA-5' and 3' untranslated regions (UTRs)-are responsible for translation efficiency. An optimal combination of the regulatory sequences allows mRNA to significantly increase the target protein's expression. We assessed the translation efficiency of mRNA encoding of firefly luciferase with various 5' and 3'UTRs in vitro on cell lines DC2.4 and THP1. We found that mRNAs containing 5'UTR sequences from eukaryotic genes HBB, HSPA1A, Rabb, or H4C2, or from the adenoviral leader sequence TPL, resulted in higher levels of luciferase bioluminescence 4 h after transfection of DC2.4 cells as compared with 5'UTR sequences used in vaccines mRNA-1273 and BNT162b2 from Moderna and BioNTech. mRNA containing TPL as the 5'UTR also showed higher efficiency (as compared with the 5'UTR from Moderna) at generating a T-cell response in mice immunized with mRNA vaccines encoding a multiepitope antigen. By contrast, no effects of various 5'UTRs and 3'UTRs were detectable in THP1 cells, suggesting that the observed effects are cell type specific. Further analyses enabled us to identify potential cell type-specific RNA-binding proteins that differ in landing sites within mRNAs with various 5'UTRs and 3'UTRs. Taken together, our data indicate high translation efficiency of TPL as a 5'UTR, according to experiments on DC2.4 cells and C57BL/6 mice.

CydDC functions as a cytoplasmic cystine reductase to sensitize Escherichia coli to oxidative stress and aminoglycosides.

l-cysteine is the source of all bacterial sulfurous biomolecules. However, the cytoplasmic level of l-cysteine must be tightly regulated due to its propensity to reduce iron and drive damaging Fenton chemistry. It has been proposed that in Escherichia coli the component of cytochrome bd-I terminal oxidase, the CydDC complex, shuttles excessive l-cysteine from the cytoplasm to the periplasm, thereby maintaining redox homeostasis. Here, we provide evidence for an alternative function of CydDC by demonstrating that the cydD phenotype, unlike that of the bona fide l-cysteine exporter eamA, parallels that of the l-cystine importer tcyP. Chromosomal induction of eamA, but not of cydDC, from a strong pLtetO-1 promoter (Ptet) leads to the increased level of extracellular l-cysteine, whereas induction of cydDC or tcyP causes the accumulation of cytoplasmic l-cysteine. Congruently, inactivation of cydD renders cells resistant to hydrogen peroxide and to aminoglycoside antibiotics. In contrast, induction of cydDC sensitizes cells to oxidative stress and aminoglycosides, which can be suppressed by eamA overexpression. Furthermore, inactivation of the ferric uptake regulator (fur) in Ptet-cydDC or Ptet-tcyP cells results in dramatic loss of survival, whereas catalase (katG) overexpression suppresses the hypersensitivity of both strains to H2O2 These results establish CydDC as a reducer of cytoplasmic cystine, as opposed to an l-cysteine exporter, and further elucidate a link between oxidative stress, antibiotic resistance, and sulfur metabolism.

Two ß-glucanases from bacterium Cellulomonas flavigena: expression in Pichia pastoris, properties, biotechnological potential.

In the genome of Cellulomonas flavigena, two genes that potentially encode endoglucanases - Cfla_2912 and Cfla_2913 were identified. We cloned the genes and created Pichia pastoris-based recombinant producers of two proteins that were expressed from the AOX1 promoter. Each of the endoglucanase molecules contains a GH6 catalytic domain, CBM2 carbohydrate-binding module, and TAT signal peptide. The fermentation of the producers was carried out in a 10 L fermenter; Cfla_2912 and Cfla_2913 were purified using affinity chromatography. The yield comprised 10.3 mg/ml (430 U/ml) for Cfla_2913 and 9 mg/ml (370 U/ml) for Cfla_2912. Cfla_2912 and Cfla_2913 were found to have a high activity against barley ß-glucan and lichenan, a weak activity against carboxymethyl cellulose (CMC), phosphoric-acid treated cellulose, and no activity against laminarin, xylan, soluble starch, microcrystalline cellulose, cellobiose, and cellotriose. Thus, the proteins exhibited ß-glucanase activity. Both proteins had a neutral pH optimum of about 7.0 and were more stable at neutral and slightly alkaline pH ranging from 7.0 to 9.0. Cfla_2912 and Cfla_2913 showed a moderate thermal stability. The products of barley ß-glucan hydrolysis by Cfla_2912 and Cfla_2913 were trisaccharide, tetrasaccharide, and cellobiose. Cfla_2912 and Cfla_2913 efficiently hydrolyzed cereal polysaccharides, which indicate that they may have biotechnological potential.

Mechanism of H2S-mediated protection against oxidative stress in Escherichia coli.

Endogenous hydrogen sulfide (H2S) renders bacteria highly resistant to oxidative stress, but its mechanism remains poorly understood. Here, we report that 3-mercaptopyruvate sulfurtransferase (3MST) is the major source of endogenous H2S in Escherichia coli Cellular resistance to H2O2 strongly depends on the activity of mstA, a gene that encodes 3MST. Deletion of the ferric uptake regulator (Fur) renders ∆mstA cells hypersensitive to H2O2 Conversely, induction of chromosomal mstA from a strong pLtetO-1 promoter (P tet -mstA) renders ∆fur cells fully resistant to H2O2 Furthermore, the endogenous level of H2S is reduced in ∆fur or ∆sodA ∆sodB cells but restored after the addition of an iron chelator dipyridyl. Using a highly sensitive reporter of the global response to DNA damage (SOS) and the TUNEL assay, we show that 3MST-derived H2S protects chromosomal DNA from oxidative damage. We also show that the induction of the CysB regulon in response to oxidative stress depends on 3MST, whereas the CysB-regulated l-cystine transporter, TcyP, plays the principle role in the 3MST-mediated generation of H2S. These findings led us to propose a model to explain the interplay between l-cysteine metabolism, H2S production, and oxidative stress, in which 3MST protects E. coli against oxidative stress via l-cysteine utilization and H2S-mediated sequestration of free iron necessary for the genotoxic Fenton reaction.

Regulation of gene expression in restriction-modification system Eco29kI.

The Eco29kI restriction-modification (R-M) system consists of two partially overlapping genes, eco29kIR, encoding a restriction endonuclease and eco29kIM, encoding methyltransferase. The two genes are thought to form an operon with the eco29kIR gene preceding the eco29kIM gene. Such an organization is expected to complicate establishment of plasmids containing this R-M system in naive hosts, since common logic dictates that methyltransferase should be synthesized first to protect the DNA from cleavage by the endonuclease. Here, we characterize the Eco29kI gene transcription. We show that a separate promoter located within the eco29kIR gene is sufficient to synthesize enough methyltransferase to completely modify host DNA. We further show that transcription from two intragenic antisense promoters strongly decreases the levels of eco29kIR gene transcripts. The antisense transcripts act by preventing translation initiation from the bicistronic eco29kIR-eco29kIM mRNA and causing its degradation. Both eco29kIM and antisense promoters are necessary for Eco29kI genes establishment and/or stable maintenance, indicating that they jointly contribute to coordinated expression of Eco29kI genes.

Transcription regulation of restriction-modification system Ecl18kI.

Restriction-modification (R-M) system Ecl18kI is representative of R-M systems whose coordinated transcription is achieved through a separate DNA-binding domain of the methyltransferase. M.Ecl18kI recognizes an operator sequence located in the noncoding region that separates the divergently transcribed R and M genes. Here we show that, contrary to previous predictions, the two ecl18kI promoters are not divergent, but actually face one another. The binding of M.Ecl18kI to its operator prevents RNA polymerase (RNAP) binding to the M promoter by steric exclusion, but has no direct effect on RNAP interaction with the R promoter. The start point for R transcription is located outside of the intergenic region, opposite the initiation codon of the M gene. Regulated transcription of the potentially toxic ecl18kI R gene is accomplished (i) at the stage of promoter complex formation, through direct competition from complexes formed at the M promoter, and (ii) at the stage of promoter clearance, since R promoter-bound RNAP escapes the promoter more slowly than RNAP bound to the M promoter.

Type II restriction endonuclease R.Eco29kI is a member of the GIY-YIG nuclease superfamily.

BACKGROUND: The majority of experimentally determined crystal structures of Type II restriction endonucleases (REases) exhibit a common PD-(D/E)XK fold. Crystal structures have been also determined for single representatives of two other folds: PLD (R.BfiI) and half-pipe (R.PabI), and bioinformatics analyses supported by mutagenesis suggested that some REases belong to the HNH fold. Our previous bioinformatic analysis suggested that REase R.Eco29kI shares sequence similarities with one more unrelated nuclease superfamily, GIY-YIG, however so far no experimental data were available to support this prediction. The determination of a crystal structure of the GIY-YIG domain of homing endonuclease I-TevI provided a template for modeling of R.Eco29kI and prompted us to validate the model experimentally. RESULTS: Using protein fold-recognition methods we generated a new alignment between R.Eco29kI and I-TevI, which suggested a reassignment of one of the putative catalytic residues. A theoretical model of R.Eco29kI was constructed to illustrate its predicted three-dimensional fold and organization of the active site, comprising amino acid residues Y49, Y76, R104, H108, E142, and N154. A series of mutants was constructed to generate amino acid substitutions of selected residues (Y49A, R104A, H108F, E142A and N154L) and the mutant proteins were examined for their ability to bind the DNA containing the Eco29kI site 5'-CCGCGG-3' and to catalyze the cleavage reaction. Experimental data reveal that residues Y49, R104, E142, H108, and N154 are important for the nuclease activity of R.Eco29kI, while H108 and N154 are also important for specific DNA binding by this enzyme. CONCLUSION: Substitutions of residues Y49, R104, H108, E142 and N154 predicted by the model to be a part of the active site lead to mutant proteins with strong defects in the REase activity. These results are in very good agreement with the structural model presented in this work and with our prediction that R.Eco29kI belongs to the GIY-YIG superfamily of nucleases. Our study provides the first experimental evidence for a Type IIP REase that does not belong to the PD-(D/E)XK or HNH superfamilies of nucleases, and is instead a member of the unrelated GIY-YIG superfamily.

Transcription regulation of the EcoRV restriction-modification system.

When a plasmid containing restriction-modification (R-M) genes enters a naïve host, unmodified host DNA can be destroyed by restriction endonuclease. Therefore, expression of R-M genes must be regulated to ensure that enough methyltransferase is produced and that host DNA is methylated before the endonuclease synthesis begins. In several R-M systems, specialized Control (C) proteins coordinate expression of the R and the M genes. C proteins bind to DNA sequences called C-boxes and activate expression of their cognate R genes and inhibit the M gene expression, however the mechanisms remain undefined. Here, we studied the regulation of gene expression in the C protein-dependent EcoRV system. We map the divergent EcoRV M and R gene promoters and we define the site of C protein-binding that is sufficient for activation of the EcoRV R transcription.

Xylanases of Cellulomonas flavigena: expression, biochemical characterization, and biotechnological potential.

Four xylanases of Cellulomonas flavigena were cloned, expressed in Escherichia coli and purified. Three enzymes (CFXyl1, CFXyl2, and CFXyl4) were from the GH10 family, while CFXyl3 was from the GH11 family. The enzymes possessed moderate temperature stability and a neutral pH optimum. The enzymes were more stable at alkaline pH values. CFXyl1 and CFXyl2 hydrolyzed xylan to form xylobiose, xylotriose, xylohexaose, xylopentaose, and xylose, which is typical for GH10. CFXyl3 (GH11) and CFXyl4 (GH10) formed the same xylooligosaccharides, but xylose was formed in small amounts. The xylanases made efficient saccharification of rye, wheat and oat, common components of animal feed, which indicates their high biotechnological potential.

Analysis of CRISPR system function in plant pathogen Xanthomonas oryzae.

Clustered regularly interspaced short palindromic repeat (CRISPR) is a bacterial immunity system that requires a perfect sequence match between the CRISPR cassette spacer and a protospacer in invading DNA for exclusion of foreign genetic elements. CRISPR cassettes are hypervariable, possibly reflecting different exposure of strains of the same species to foreign genetic elements. Here, we determined CRISPR cassette sequences of two Xanthomonas oryzae strains and found that one of the strains remains sensitive to phage Xop411 despite carrying a cassette that has a spacer exactly matching a fragment of the Xop411 genome. To explain this apparent paradox, we identified X. oryzae CRISPR spacers of likely phage origin and defined a consensus sequence of a motif adjacent to X. oryzae phage protospacers. Our analysis revealed that the Xop411 protospacer that matches the CRISPR spacer has this motif mutated, which likely explains the phage's ability to infect its host. While similar observations were made previously with Streptococcus thermophilus and its phages, the conserved motif in X. oryzae phages is located on a protospacer side opposite to the S. thermophilus phages' motif. The results thus point to a considerable degree of variety of CRISPR-mediated phage resistance mechanisms in different bacteria.