Whole-genome sequence of Bacillus velezensis strain R22 isolated from Oryza sativa rhizosphere in Bulgaria.

Bacillus velezensis R22 was isolated from a rice rhizosphere in Bulgaria. Its genome (assembled into 14 scaffolds) has a size of 4.08 Mbp and a G + C content of 46.35%. Nine full biosynthetic clusters for antimicrobials were predicted, among them two new gene clusters probably encoding polyketides named macrolactin R22 and velezensin.

Influence of pH on Inulin Conversion to 2,3-Butanediol by Bacillus licheniformis 24: A Gene Expression Assay.

2,3-Butanediol (2,3-BD) is an alcohol highly demanded in the chemical, pharmaceutical, and food industries. Its microbial production, safe non-pathogenic producer strains, and suitable substrates have been avidly sought in recent years. The present study investigated 2,3-BD synthesis by the GRAS Bacillus licheniformis 24 using chicory inulin as a cheap and renewable substrate. The process appears to be pH-dependent. At pH 5.25, the synthesis of 2,3-BD was barely detectable due to the lack of inulin hydrolysis. At pH 6.25, 2,3-BD concentration reached 67.5 g/L with rapid hydrolysis of the substrate but was accompanied by exopolysaccharide (EPS) synthesis. Since inulin conversion by bacteria is a complex process and begins with its hydrolysis, the question of the acting enzymes arose. Genome mining revealed that several glycoside hydrolase (GH) enzymes from different CAZy families are involved. Five genes encoding such enzymes in B. licheniformis 24 were amplified and sequenced: sacA, sacB, sacC, levB, and fruA. Real-time RT-PCR experiments showed that the process of inulin hydrolysis is regulated at the level of gene expression, as four genes were significantly overexpressed at pH 6.25. In contrast, the expression of levB remained at the same level at the different pH values at all-time points. It was concluded that the sacC and sacA/fruA genes are crucial for inulin hydrolysis. They encode exoinulinase (EC 3.2.1.80) and sucrases (EC 3.2.1.26), respectively. The striking overexpression of sacB under these conditions led to increased synthesis of EPS; therefore, the simultaneous production of 2,3-BD and EPS cannot be avoided.

Multiple cry Genes in Bacillus thuringiensis Strain BTG Suggest a Broad-Spectrum Insecticidal Activity.

The properties of Bacillus thuringiensis strains as a biopesticide with potent action against moths, beetles, and mosquitoes have been known for decades, with individual subspecies showing specific activity against a particular pest. The aim of the present work is to characterize strains that can be used for broad-spectrum pest control in agriculture. Twenty strains of B. thuringiensis were isolated from Bulgarian soil habitats. The strains were screened for genes encoding 12 different crystal (Cry) endotoxins by PCR with specific primer pairs. Seven of the isolates contained cry genes in their genomes. B. thuringiensis strains PL1, PL3, and PL20 contained at least three different cry genes, while B. thuringiensis serovar galleriae BTG contained at least four. Moreover, scanning electron microscopy (SEM) investigation revealed the production of bipyramidal (PL1, PL3, PL20), polygonal (PL1), cubic (BTG), and spherical crystals (BTG and PL20). Potentially containing the most cry genes, the BTG genome was sequenced and annotated. It comprises 6,275,416 base pairs, does not contain plasmids, has a GC content of 35.05%, and contained 7 genes encoding crystal toxins: cry1Ab35, cry1Db, cry1Fb, cry1Ib, cry2Ab, cry8Ea1, and cry9Ba. This unique combination would possibly enable the simultaneous pesticidal action against pest species from orders Lepidoptera, Coleoptera, Diptera, and Hemiptera, as well as class Gastropoda. Whole-genome sequencing provided accurate information about the presence, localization, and classification of Cry toxins in B. thuringiensis BTG, revealing the great potential of the strain for the development of new broad-spectrum bio-insecticides.

Microbial Detoxification of Residual Pesticides in Fermented Foods: Current Status and Prospects.

The treatment of agricultural areas with pesticides is an indispensable approach to improve crop yields and cannot be avoided in the coming decades. At the same time, significant amounts of pesticides remain in food and their ingestion causes serious damage such as neurological, gastrointestinal, and allergic reactions; cancer; and even death. However, during the fermentation processing of foods, residual amounts of pesticides are significantly reduced thanks to enzymatic degradation by the starter and accompanying microflora. This review concentrates on foods with the highest levels of pesticide residues, such as milk, yogurt, fermented vegetables (pickles, kimchi, and olives), fruit juices, grains, sourdough, and wines. The focus is on the molecular mechanisms of pesticide degradation due to the presence of specific microbial species. They contain a unique genetic pool that confers an appropriate enzymological profile to act as pesticide detoxifiers. The prospects of developing more effective biodetoxification strategies by engaging probiotic lactic acid bacteria are also discussed.

Safe Sialidase Production by the Saprophyte Oerskovia paurometabola: Gene Sequence and Enzyme Purification.

Sialidase preparations are applied in structural and functional studies on sialoglycans, in the production of sialylated therapeutic proteins and synthetic substrates for use in biochemical research, etc. They are obtained mainly from pathogenic microorganisms; therefore, the demand for apathogenic producers of sialidase is of exceptional importance for the safe production of this enzyme. Here, we report for the first time the presence of a sialidase gene and enzyme in the saprophytic actinomycete Oerskovia paurometabola strain O129. An electrophoretically pure, glycosylated enzyme with a molecular weight of 70 kDa was obtained after a two-step chromatographic procedure using DEAE cellulose and Q-sepharose. The biochemical characterization showed that the enzyme is extracellular, inductive, and able to cleave α(2→3,6,8) linked sialic acids with preference for α(2→3) bonds. The enzyme production was strongly induced by glycomacropeptide (GMP) from milk whey, as well as by sialic acid. Investigation of the deduced amino acid sequence revealed that the protein molecule has the typical six-bladed ß-propeller structure and contains all features of bacterial sialidases, i.e., an YRIP motif, five Asp-boxes, and the conserved amino acids in the active site. The presence of an unusual signal peptide of 40 amino acids was predicted. The sialidase-producing O. paurometabola O129 showed high and constant enzyme production. Together with its saprophytic nature, this makes it a reliable producer with high potential for industrial application.

In Vitro Production of Galactooligosaccharides by a Novel ß-Galactosidase of Lactobacillus bulgaricus.

ß-galactosidase is an enzyme with dual activity and important industrial application. As a hydrolase, the enzyme eliminates lactose in milk, while as a trans-galactosidase it produces prebiotic galactooligosaccharides (GOS) with various degrees of polymerization (DP). The aim of the present study is the molecular characterization of ß-galactosidase from a Bulgarian isolate, Lactobacillus delbrueckii subsp. bulgaricus 43. The sequencing of the ß-gal gene showed that it encodes a new enzyme with 21 amino acid replacements compared to all other ß-galactosidases of this species. The molecular model revealed that the new ß-galactosidase acts as a tetramer. The amino acids D207, H386, N464, E465, Y510, E532, H535, W562, N593, and W980 form the catalytic center and interact with Mg2+ ions and substrate. The ß-gal gene was cloned into a vector allowing heterologous expression of E. coli BL21(DE3) with high efficiency, as the crude enzyme reached 3015 U/mL of the culture or 2011 U/mg of protein. The enzyme's temperature optimum at 55 °C, a pH optimum of 6.5, and a positive influence of Mg2+, Mn2+, and Ca2+ on its activity were observed. From lactose, ß-Gal produced a large amount of GOS with DP3 containing ß-(1→3) and ß-(1→4) linkages, as the latter bond is particularly atypical for the L. bulgaricus enzymes. DP3-GOS formation was positively affected by high lactose concentrations. The process of lactose conversion was rapid, with a 34% yield of DP3-GOS in 6 h, and complete degradation of 200 g/L of lactose for 12 h. On the other hand, the enzyme was quite stable at 55 °C and retained about 20% of its activity after 24 h of incubation at this temperature. These properties expand our horizons as regards the use of ß-galactosidases in industrial processes for the production of lactose-free milk and GOS-enriched foods.

The Complex Role of Lactic Acid Bacteria in Food Detoxification.

Toxic ingredients in food can lead to serious food-related diseases. Such compounds are bacterial toxins (Shiga-toxin, listeriolysin, Botulinum toxin), mycotoxins (aflatoxin, ochratoxin, zearalenone, fumonisin), pesticides of different classes (organochlorine, organophosphate, synthetic pyrethroids), heavy metals, and natural antinutrients such as phytates, oxalates, and cyanide-generating glycosides. The generally regarded safe (GRAS) status and long history of lactic acid bacteria (LAB) as essential ingredients of fermented foods and probiotics make them a major biological tool against a great variety of food-related toxins. This state-of-the-art review aims to summarize and discuss the data revealing the involvement of LAB in the detoxification of foods from hazardous agents of microbial and chemical nature. It is focused on the specific properties that allow LAB to counteract toxins and destroy them, as well as on the mechanisms of microbial antagonism toward toxigenic producers. Toxins of microbial origin are either adsorbed or degraded, toxic chemicals are hydrolyzed and then used as a carbon source, while heavy metals are bound and accumulated. Based on these comprehensive data, the prospects for developing new combinations of probiotic starters for food detoxification are considered.

New Exopolysaccharides Produced by Bacillus licheniformis 24 Display Substrate-Dependent Content and Antioxidant Activity.

Bacillus licheniformis is a soil bacterium with many industrial applications. In addition to enzymes, platform chemicals, antibiotics and phytohormones, the species produces exopolysaccharides (EPSs) of various biological activities. This study revealed that Bulgarian isolate B. licheniformis 24 produced EPSs consisting of galactose, glucose and mannose with substrate-dependent ratio. From glucose, B. licheniformis 24 secreted EPS1, consisting of 54% galactose, 39% glucose and 7% mannose. From fructose, the strain formed EPS2, containing 51% glucose, 30% mannose and 19% galactose. Batch cultivation in flasks yielded 2.2-2.6 g/L EPS1 and 1.90-2.11 g/L EPS2. Four to five times higher yields of EPS were obtained from both substrates during batch and fed-batch processes in a fermenter at 37.8 °C, pH 6.2 and aeration 3.68 vvm. The batch process with 200 g/L of starting substrates received 9.64 g/L EPS1 and 6.29 g/L EPS2, reaching maximum values at the 33rd and 24th h, respectively. Fed-batch fermentation resulted in the highest yields, 12.61 g/L EPS1 and 7.03 g/L EPS2. In all processes, EPSs were produced only in the exponential growth phase. Both EPSs exhibited antioxidant activity, but EPS2 was much more potent in this regard, reaching 811 µM Vitamin C Equivalent Antioxidant Capacity (versus 135 µM for EPS1). EPS1 displayed antibacterial activity against a non-O1 strain of Vibrio cholerae.

Enhanced Activity by Genetic Complementarity: Heterologous Secretion of Clostridial Cellulases by Bacillus licheniformis and Bacillus velezensis.

To adapt to various ecological niches, the members of genus Bacillus display a wide spectrum of glycoside hydrolases (GH) responsible for the hydrolysis of cellulose and lignocellulose. Being abundant and renewable, cellulose-containing plant biomass may be applied as a substrate in second-generation biotechnologies for the production of platform chemicals. The present study aims to enhance the natural cellulase activity of two promising 2,3-butanediol (2,3-BD) producers, Bacillus licheniformis 24 and B. velezensis 5RB, by cloning and heterologous expression of cel8A and cel48S genes of Acetivibrio thermocellus. In B. licheniformis, the endocellulase Cel8A (GH8) was cloned to supplement the action of CelA (GH9), while in B. velezensis, the cellobiohydrolase Cel48S (GH48) successfully complemented the activity of endo-cellulase EglS (GH5). The expression of the natural and heterologous cellulase genes in both hosts was demonstrated by reverse-transcription PCR. The secretion of clostridial cellulases was additionally enhanced by enzyme fusion to the subtilisin-like signal peptide, reaching a significant increase in the cellulase activity of the cell-free supernatants. The results presented are the first to reveal the possibility of genetic complementation for enhancement of cellulase activity in bacilli, thus opening the prospect for genetic improvement of strains with an important biotechnological application.

Traditional Bulgarian Dairy Products: Ethnic Foods with Health Benefits.

The reported health effects of fermented dairy foods, which are traditionally manufactured in Bulgaria, are connected with their microbial biodiversity. The screening and development of probiotic starters for dairy products with unique properties are based exclusively on the isolation and characterization of lactic acid bacterial (LAB) strains. This study aims to systematically describe the LAB microbial content of artisanal products such as Bulgarian-type yoghurt, white brined cheese, kashkaval, koumiss, kefir, katak, and the Rhodope's brano mliako. The original technologies for their preparation preserve the valuable microbial content and improve their nutritional and probiotic qualities. This review emphasises the features of LAB starters and the autochthonous microflora, the biochemistry of dairy food production, and the approaches for achieving the fortification of the foods with prebiotics, bioactive peptides (ACE2-inhibitors, bacteriocins, cyclic peptides with antimicrobial activity), immunomodulatory exopolysaccharides, and other metabolites (indol-3-propionic acid, free amino acids, antioxidants, prebiotics) with reported beneficial effects on human health. The link between the microbial content of dairy foods and the healthy human microbiome is highlighted.

Butanol Tolerance of Lactiplantibacillus plantarum: A Transcriptome Study.

Biobutanol is a promising alternative fuel with impaired microbial production thanks to its toxicity. Lactiplantibacillus plantarum (L. plantarum) is among the few bacterial species that can naturally tolerate 3% (v/v) butanol. This study aims to identify the genetic factors involved in the butanol stress response of L. plantarum by comparing the differential gene expression in two strains with very different butanol tolerance: the highly resistant Ym1, and the relatively sensitive 8-1. During butanol stress, a total of 319 differentially expressed genes (DEGs) were found in Ym1, and 516 in 8-1. Fifty genes were upregulated and 54 were downregulated in both strains, revealing the common species-specific effects of butanol stress: upregulation of multidrug efflux transporters (SMR, MSF), toxin-antitoxin system, transcriptional regulators (TetR/AcrR, Crp/Fnr, and DeoR/GlpR), Hsp20, and genes involved in polysaccharide biosynthesis. Strong inhibition of the pyrimidine biosynthesis occurred in both strains. However, the strains differed greatly in DEGs responsible for the membrane transport, tryptophan synthesis, glycerol metabolism, tRNAs, and some important transcriptional regulators (Spx, LacI). Uniquely upregulated in the butanol-resistant strain Ym1 were the genes encoding GntR, GroEL, GroES, and foldase PrsA. The phosphoenolpyruvate flux and the phosphotransferase system (PTS) also appear to be major factors in butanol tolerance.

How to outwit nature: Omics insight into butanol tolerance.

The energy crisis, depletion of oil reserves, and global climate changes are pressing problems of developed societies. One possibility to counteract that is microbial production of butanol, a promising new fuel and alternative to many petrochemical reagents. However, the high butanol toxicity to all known microbial species is the main obstacle to its industrial implementation. The present state of the art review aims to expound the recent advances in modern omics approaches to resolving this insurmountable to date problem of low butanol tolerance. Genomics, transcriptomics, and proteomics show that butanol tolerance is a complex phenomenon affecting multiple genes and their expression. Efflux pumps, stress and multidrug response, membrane transport, and redox-related genes are indicated as being most important during butanol challenge, in addition to fine-tuning of global regulators of transcription (Spo0A, GntR), which may further improve tolerance. Lipidomics shows that the alterations in membrane composition (saturated lipids and plasmalogen increase) are very much species-specific and butanol-related. Glycomics discloses the pleiotropic effect of CcpA, the role of alternative sugar transport, and the production of exopolysaccharides as alternative routes to overcoming butanol stress. Unfortunately, the strain that simultaneously syntheses and tolerates butanol in concentrations that allow its commercialization has not yet been discovered or produced. Omics insight will allow the purposeful increase of butanol tolerance in natural and engineered producers and the effective heterologous expression of synthetic butanol pathways in strains hereditary butanol-resistant up to 3.2 - 4.9% (w/v). Future breakthrough can be achieved by a detailed study of the membrane proteome, of which 21% are proteins with unknown functions.

Long-term cyclic strain downregulates endothelial Nox4.

Endothelial cells in vivo are constantly exposed to mechanical forces such as cyclic strain. In endothelial cells, Nox4-containing NAD(P)H oxidase complexes have been identified as major sources of superoxide anion (.O(2)(-)) formation. In this study, we analyzed the effect of cyclic strain on endothelial ROS formation by electron paramagnetic resonance spectroscopy, cytochrome c assay, and dihydroethidium fluorescence, on NO formation by Griess reaction and on gene expression by RT-PCR and Western blot. Primary cultures of human umbilical vein endothelial cells were exposed to 2-18% cyclic strain for up to 24 h using the Flexercell system. Long-term application of 5-12% cyclic strain downregulated Nox4 expression and ROS formation in a time-dependent manner. Downregulation of Nox4 was further confirmed by promoter analysis using dual-luciferase assay. Cu/Zn SOD, MnSOD, and catalase expression was decreased after application of chronic 12% cyclic strain. In contrast, endothelial NO formation and eNOS were increased by cyclic strain. Strain-dependent Nox4 downregulation was abolished by eNOS inhibition with L-NAME. In conclusion, physiological levels of cyclic strain downregulate Nox4 expression and superoxide anion formation. This novel mechanism might contribute to a vasoprotective balance between NO and superoxide anions in response to physiological mechanical stimulation of endothelial cells.