Accurate noise-robust classification of Bacillus species from MALDI-TOF MS spectra using a denoising autoencoder.

Bacillus strains are ubiquitous in the environment and are widely used in the microbiological industry as valuable enzyme sources, as well as in agriculture to stimulate plant growth. The Bacillus genus comprises several closely related groups of species. The rapid classification of these remains challenging using existing methods. Techniques based on MALDI-TOF MS data analysis hold significant promise for fast and precise microbial strains classification at both the genus and species levels. In previous work, we proposed a geometric approach to Bacillus strain classification based on mass spectra analysis via the centroid method (CM). One limitation of such methods is the noise in MS spectra. In this study, we used a denoising autoencoder (DAE) to improve bacteria classification accuracy under noisy MS spectra conditions. We employed a denoising autoencoder approach to convert noisy MS spectra into latent variables representing molecular patterns in the original MS data, and the Random Forest method to classify bacterial strains by latent variables. Comparison of the DAE-RF with the CM method using the artificially noisy test samples showed that DAE-RF offers higher noise robustness. Hence, the DAE-RF method could be utilized for noise-robust, fast, and neat classification of Bacillus species according to MALDI-TOF MS data.

Heterologous Expression of Extracellular Proteinase pAsPs of Aspergillus pseudotamarii in Komagataella phaffii.

Neutral protease pAsPs gene was obtained by sequence optimization of NpI protease from Aspergillus pseudotamarii. pAsPs was for the first time integrated in the genome of yeast strain Komagataella phaffii T07, and then produced in a 5 L bioreactor with an enzyme yield of 150,800 U/mL of culture liquid towards casein. The specific activity of the pAsPs was 7,657,000 U/mg toward casein, 2320 U/mg toward hemoglobin, and 25,344 U/mg toward azocasein per 1 mg of the protein. The enzyme was found to be inhibited by Cu2+. Optimal activity pH was shown in the range of pH 6.5-8.0, and optimal temperature-50-60 °C. The molecular mass of the recombinant protease pAsPs was shown to be 67.5 kDa. Mass-spectrometric analysis confirmed the identity of the amino acid sequence of the obtained pAsPs preparation with the predicted sequence, with 17% coverage and protein score 288. Thus, the novel neutral protease pAsPs is a promising candidate for large-scale use in manufacturing, including the food industry.

Specific Features of the Proteomic Response of Thermophilic Bacterium Geobacillus icigianus to Terahertz Irradiation.

Studying the effects of terahertz (THz) radiation on the proteome of temperature-sensitive organisms is limited by a number of significant technical difficulties, one of which is maintaining an optimal temperature range to avoid thermal shock as much as possible. In the case of extremophilic species with an increased temperature tolerance, it is easier to isolate the effects of THz radiation directly. We studied the proteomic response to terahertz radiation of the thermophilic Geobacillus icigianus, persisting under wide temperature fluctuations with a 60 °C optimum. The experiments were performed with a terahertz free-electron laser (FEL) from the Siberian Center for Synchrotron and Terahertz Radiation, designed and employed by the Institute of Nuclear Physics of the SB of the RAS. A G. icigianus culture in LB medium was THz-irradiated for 15 min with 0.23 W/cm2 and 130 µm, using a specially designed cuvette. The life cycle of this bacterium proceeds under conditions of wide temperature and osmotic fluctuations, which makes its enzyme systems stress-resistant. The expression of several proteins was shown to change immediately after fifteen minutes of irradiation and after ten minutes of incubation at the end of exposure. The metabolic systems of electron transport, regulation of transcription and translation, cell growth and chemotaxis, synthesis of peptidoglycan, riboflavin, NADH, FAD and pyridoxal phosphate cofactors, Krebs cycle, ATP synthesis, chaperone and protease activity, and DNA repair, including methylated DNA, take part in the fast response to THz radiation. When the response developed after incubation, the systems of the cell's anti-stress defense, chemotaxis, and, partially, cell growth were restored, but the respiration and energy metabolism, biosynthesis of riboflavin, cofactors, peptidoglycan, and translation system components remained affected and the amino acid metabolism system was involved.

Heterologous Expression of Xylanase xAor from Aspergillus oryzae in Komagataella phaffii T07

Xylanases (EC 3.2.1.8) hydrolyze the hemicellulose of plant cell walls. Xylanases are used in the food and paper industries and for bioconversion of lignocellulose to biofuel. In this work, the producer-strain with four copies of the xAor xylanase gene was organized in two tandem copies for optimal expression in Komagataella phaffii T07 yeast. The secreted 35 kDa xylanase was purified from culture medium by gel filtration on Sephadex G-25 and anion exchange chromatography on DEAE-Sepharose 6HF. Tryptic peptides of the recombinant enzyme were analyzed by liquid chromatography-tandem mass spectrometry where the amino acid sequence corresponded to Protein Accession # O94163 for Endo-1,4-beta-xylanase from Aspergillus oryzae RIB40. The recombinant xylanase was produced in a bioreactor where the secreted enzyme hydrolyzed oat xylane with an activity of 258240 IU/mL. High activity in the culture medium suggested xylanase could be used for industrial applications without being purified or concentrated. The pH optimum for xylanase xAor was 7.5, though the enzyme was active from pH 2.5 to pH 10. Xylanase was active at temperatures from 35 °C to 85 °C with a maximum at 60 °C. In conclusion, this protocol yields soluble, secreted xylanase suitable for industrial scale production.

E. coli aggregation and impaired cell division after terahertz irradiation.

In this study we demonstrated that exposure of Escherichia coli (E. coli) to terahertz (THz) radiation resulted in a change in the activities of the tdcABCDEFGR and matA-F genes (signs of cell aggregation), gene yjjQ (signs of suppression of cell motility), dicABCF, FtsZ, and minCDE genes (signs of suppression of cell division), sfmACDHF genes (signs of adhesin synthesis), yjbEFGH and gfcA genes (signs of cell envelope stabilization). Moreover, THz radiation induced E. coli csg operon genes of amyloid biosynthesis. Electron microscopy revealed that the irradiated bacteria underwent increased aggregation; 20% of them formed bundle-like structures consisting of two to four pili clumped together. This could be the result of changes in the adhesive properties of the pili. We also found aberrations in cell wall structure in the middle part of the bacterial cell; these aberrations impaired the cell at the initial stages of division and resulted in accumulation of long rod-like cells. Overall, THz radiation was shown to have adverse effects on bacterial populations resulting in cells with abnormal morphology.

Study on the effects of terahertz radiation on gene networks of Escherichia coli by means of fluorescent biosensors.

Three novel fluorescent biosensors sensitive to terahertz (THz) radiation were developed via transformation of Escherichia coli (E. coli) cells with plasmids, in which a promotor of genes matA, safA, or chbB controls the expression of a fluorescent protein. The biosensors were exposed to THz radiation from two sources: a high-intensity pulsed short-wave free electron laser and a low-intensity continuous long-wave IMPATT-diode-based device. The threshold and dynamics of fluorescence were found to depend on radiation parameters and exposure time. Heat shock or chemical stress yielded the absence of fluorescence induction. The biosensors are evaluated to be suitable for studying influence of THz radiation on the activity of gene networks related with considered gene promoters.

The recombinant fusion protein CFP10-ESAT6-dIFN has protective effect against tuberculosis in guinea pigs.

Development of effective vaccine candidates against tuberculosis is currently the most important challenge in the prevention of this disease since the BCG vaccine fails to guarantee a lifelong protection, while any other approved vaccine with better efficiency is still absent. The protective effect of the recombinant fusion protein ESAT6-CFP10-dIFN produced in a prokaryotic expression system (Escherichia coli) has been assessed in a guinea pig model of acute tuberculosis. The tested antigen comprises the Mycobacterium tuberculosis (Mtb) proteins ESAT6 and CFP10 as well as modified human γ-interferon (dIFN) for boosting the immune response. Double intradermal immunization of animals with the tested fusion protein (2 × 0.5 µg) induces a protective effect against subsequent Mtb infection. The immunized animals do not develop the symptoms of acute tuberculosis and their body weight gain was five times more as compared with the non-immunized-infected animals. The animal group immunized with this dose of antigen displays the minimum morphological changes in the internal organs and insignificant inflammatory lesions in the liver tissue, which complies with a decrease in the bacterial load in the spleen and average Mtb counts in macrophages.

The recombinant fusion protein CFP10-ESAT6-dIFN has protective effect against tuberculosis in guinea pigs.

Development of effective vaccine candidates against tuberculosis (TB) is currently the most important challenge in the prevention of this disease since the BCG vaccine fails to guarantee a lifelong protection, while any other approved vaccine with better efficiency is still absent. The protective effect of the recombinant fusion protein CFP10-ESAT6-dIFN produced in a prokaryotic expression system (Escherichia coli) has been assessed in a guinea pig model of acute TB. The tested antigen comprises the Mycobacterium tuberculosis (Mtb) proteins ESAT6 and CFP10 as well as modified human γ-interferon (dIFN) for boosting the immune response. Double intradermal immunization of guinea pigs with the tested fusion protein (2 × 0.5 µg) induces a protective effect against subsequent Mtb infection. The immunized guinea pigs do not develop the symptoms of acute TB and their body weight gain was five times more as compared with the non-immunized infected guinea pigs. The animal group immunized with this dose of antigen displays the minimum morphological changes in the internal organs and insignificant inflammatory lesions in the liver tissue, which complies with a decrease in the bacterial load in the spleen and average Mtb counts in macrophages.

Studying the non-thermal effects of terahertz radiation on E. coli/pKatG-GFP biosensor cells.

Studies of the impact of terahertz radiation on living objects present a significant interest since its use for security systems is currently considered promising. We studied the non-thermal impact of terahertz radiation on E. coli/pKatG-gfp biosensor cells. The Novosibirsk free electron laser (NovoFEL), which currently has the world's highest average and peak power, was used as the source of terahertz radiation. We demonstrated that exposure to terahertz radiation at the wavelengths of 130, 150, and 200 µm and a power of 1.4 W/cm(2) induces changes in green fluorescent protein (GFP) fluorescence values and thus induces the expression of GFP in E. coli/pKatG-gfp biosensor cells. Possible mechanisms of the E. coli response to non-thermal exposure to terahertz radiation are discussed.

Stability and association with the cytomatrix of mitochondrial DNA in spontaneously immortalized mouse embryo fibroblasts containing or lacking the intermediate filament protein vimentin.

To extend previous observations demonstrating differences in number, morphology, and activity of mitochondria in spontaneously immortalized vim(+) and vim(-) fibroblasts derived from wild-type and vimentin knockout mice, some structural and functional aspects of mitochondrial genome performance and integrity in both types of cells were investigated. Primary Vim(+/+) and Vim(-/-) fibroblasts, which escaped terminal differentiation by immortalization were characterized by an almost twofold lower mtDNA content in comparison to that of their primary precursor cells, whereby the average mtDNA copy number in two clones of vim(+) cells was lower by a factor of 0.6 than that in four clones of vim(-) cells. However, during serial subcultivation up to high passage numbers, the vim(+) and vim() fibroblasts increased their mtDNA copy number 1.5- and 2.5-fold, respectively. While early-passage cells of the vim(+) and vim(-) fibroblast clones differed only slightly in the ratio between mtDNA content and mitochondrial mass represented by mtHSP70 protein, after ca. 300 population doublings the average mtDNA/mtmass ratio in the vim(+) and vim() cells was increased by a factor of 2 and 4.5, respectively. During subcultivation, both types of cells acquired the fully transformed phenotype. These findings suggest that cytoskeletal vimentin filaments exert a strong influence on the mechanisms controlling mtDNA copy number during serial subcultivation of immortalized mouse embryo fibroblasts, and that vimentin deficiency causes a disproportionately enhanced mtDNA content in high-passage vim(-) fibroblasts. Such a role of vimentin filaments was supported by the stronger retention potential for mtDNA and mtDNA polymerase (gamma) detected in vim(+) fibroblasts by Triton X-100 extraction of mitochondria and agaroseembedded cells. Moreover, although the vim(+) and vim(-) fibroblasts were equally active in generating free radicals, the vim(-) cells exhibited higher levels of immunologically detectable 8-oxoG and mismatch repair proteins MSH2 and MLH1 in their mitochondria. Because in vim(-) fibroblasts only one point mutation was detected in the mtDNA D-loop control region, these cells are apparently able to efficiently remove oxidatively damaged nucleobases. On the other hand, a number of large-scale mtDNA deletions were found in high-passage vim(-) fibroblasts, but not in low-passage vim(-) cells and vim(+) cells of both low and high passage. Large mtDNA deletions were also induced in young vim(-) fibroblasts by treatment with the DNA intercalator ethidium bromide, whereas no such deletions were found after treatment of vim(+) cells. These results indicate that in immortalized vim(-) fibroblasts the mitochondrial genome is prone to large-scale rearrangements, probably due to insufficient control of mtDNA repair and recombination processes in the absence of vimentin.