The Role of Probiotics and Their Metabolites in the Treatment of Depression.

Depression is a common and complex mental and emotional disorder that causes disability, morbidity, and quite often mortality around the world. Depression is closely related to several physical and metabolic conditions causing metabolic depression. Studies have indicated that there is a relationship between the intestinal microbiota and the brain, known as the gut-brain axis. While this microbiota-gut-brain connection is disturbed, dysfunctions of the brain, immune system, endocrine system, and gastrointestinal tract occur. Numerous studies show that intestinal dysbiosis characterized by abnormal microbiota and dysfunction of the microbiota-gut-brain axis could be a direct cause of mental and emotional disorders. Traditional treatment of depression includes psychotherapy and pharmacotherapy, and it mainly targets the brain. However, restoration of the intestinal microbiota and functions of the gut-brain axis via using probiotics, their metabolites, prebiotics, and healthy diet may alleviate depressive symptoms. Administration of probiotics labeled as psychobiotics and their metabolites as metabiotics, especially as an adjuvant to antidepressants, improves mental disorders. It is a new approach to the prevention, management, and treatment of mental and emotional illnesses, particularly major depressive disorder and metabolic depression. For the effectiveness of antidepressant therapy, psychobiotics should be administered at a dose higher than 1 billion CFU/day for at least 8 weeks.

Response of murine gut microbiota to a prebiotic based on oligosaccharides derived via hydrolysis of fungal α-(1→3)-d-glucan: Preclinical trial study on mice.

We investigated the modulating effect of α-(1→3)-glucooligosaccharides (GOS), i.e. a product of fungal α-(1→3)-d-glucan hydrolysis, on the gut microbiota composition. Mice were fed with a GOS-supplemented diet and two control diets for 21 days, and fecal samples were collected at 0, 1, and 3-week time points. The bacterial community composition was determined by 16S rRNA gene Illumina sequencing. The gut microbiota of the GOS-supplemented mice showed profound time-dependent changes in the taxonomic composition; however, we did not observe significant changes in α-diversity indices. The biggest number of genus abundance shifts after 1 week of the treatment was noticed between the group of the GOS-supplemented mice and the controls; however, the differences were still relevant after the 3-week treatment. The GOS-supplemented mice displayed higher abundance of Prevotella spp., with a concomitant decrease in the abundance of Escherichia-Shigella. Hence, GOS seems to be a promising candidate for a new prebiotic.

Utilization of Legume-Nodule Bacterial Symbiosis in Phytoremediation of Heavy Metal-Contaminated Soils.

With the increasing industrial activity of the growing human population, the accumulation of various contaminants in soil, including heavy metals, has increased rapidly. Heavy metals as non-biodegradable elements persist in the soil environment and may pollute crop plants, further accumulating in the human body causing serious conditions. Hence, phytoremediation of land contamination as an environmental restoration technology is desirable for both human health and broad-sense ecology. Legumes (Fabaceae), which play a special role in nitrogen cycling, are dominant plants in contaminated areas. Therefore, the use of legumes and associated nitrogen-fixing rhizobia to reduce the concentrations or toxic effects of contaminants in the soil is environmentally friendly and becomes a promising strategy for phytoremediation and phytostabilization. Rhizobia, which have such plant growth-promoting (PGP) features as phosphorus solubilization, phytohormone synthesis, siderophore release, production of beneficial compounds for plants, and most of all nitrogen fixation, may promote legume growth while diminishing metal toxicity. The aim of the present review is to provide a comprehensive description of the main effects of metal contaminants in nitrogen-fixing leguminous plants and the benefits of using the legume-rhizobium symbiosis with both wild-type and genetically modified plants and bacteria to enhance an efficient recovery of contaminated lands.

Comparative Nanopore Sequencing-Based Evaluation of the Midgut Microbiota of the Summer Chafer (Amphimallon solstitiale L.) Associated with Possible Resistance to Entomopathogenic Nematodes.

Root-feeding Amphimallon solstitiale larvae and certain other scarab beetles are the main soil-dwelling pests found in Europe, while entomopathogenic nematodes (EPN) have been used as a biocontrol agent against these species. Our study provides the first detailed characterization of the bacterial community of the midgut in wild A. solstitiale larvae, based on the nanopore sequencing of the 16S rRNA gene. In the whole dataset, we detected 2586 different genera and 11,641 species, with only 83 diverse bacterial genera shared by all studied individuals, which may represent members of the core midgut microbiota of A. solstitiale larvae. Subsequently, we compared the midgut microbiota of EPN-resistant and T0 (prior to EPN exposure) individuals, hypothesizing that resistance to this parasitic infection may be linked to the altered gut community. Compared to the control, the resistant insect microbiota demonstrated lower Shannon and Evenness indices and significant differences in the community structure. Our studies confirmed that the gut microbiota alternation is associated with resistant insects; however, there are many processes involved that can affect the bacterial community. Further research on the role of gut microbiota in insect-parasitic nematode interaction may ultimately lead to the improvement of biological control strategies in insect pest management.

Screening and Molecular Identification of Bacteria from the Midgut of Amphimallon solstitiale Larvae Exhibiting Antagonistic Activity against Bacterial Symbionts of Entomopathogenic Nematodes.

Entomopathogenic nematodes (Rhabditida: Steinernematidae and Heterorhabditidae) are a group of organisms capable of infecting larvae of insects living in soil, including representatives of the family Scarabaeidae. Their insecticidal activity is related to the presence of symbiotic bacteria Xenorhabdus spp. or Photorhabdus spp. in the alimentary tract, which are released into the insect body, leading to its death caused by bacterial toxins and septicemia. Although the antibacterial activities of symbionts of entomopathogenic nematodes have been well described, there is insufficient knowledge of the interactions between these bacteria and microorganisms that naturally inhabit the alimentary tract of insects infested by nematodes. In this study, 900 bacterial strains isolated from midgut samples of Amphimallon solstitiale larvae were tested for their antagonistic activity against the selected five Xenorhabdus and Photorhabdus species. Cross-streak tests showed significant antibacterial activity of 20 isolates. These bacteria were identified as Bacillus [Brevibacterium] frigoritolerans, Bacillus toyonensis, Bacillus wiedmannii, Chryseobacterium lathyri, Chryseobacterium sp., Citrobacter murliniae, Enterococcus malodoratus, Paenibacillus sp., Serratia marcescens and Serratia sp. Since some representatives of the intestinal microbiota of A. solstitiale are able to inhibit the growth of Xenorhabdus and Photorhrhabdus bacteria in vitro, it can be assumed that this type of bacterial interaction may occur at certain stages of insect infection by Steinernema or Heterorhabditis nematodes.

Steinernema sandneri n. sp. (Rhabditida: Steinernematidae), a new entomopathogenic nematode from Poland.

A new species of entomopathogenic nematodes, Steinernema sandneri n. sp., was recovered by baiting from Poland. Its morphological traits indicate that the new species is a member of the feltiae-kraussei group. A body length of 843 (708-965) µm, a more anterior position of excretory pore (56 µm), and the lower D% value (40 vs > 46) discriminate this species from most of the other group members. The first-generation males of S. sandneri n. sp. can be distinguished from the other clade members by a 60 µm long spicule, a relatively long gubernaculum (GS% = 79), and the position of the excretory pore (80 µm). Phylogenetic analysis of the ITS rDNA, D2D3 of 28 S rDNA, and cox1 sequences confirmed that S. sandneri n. sp. is a new species of the feltiae-kraussei group, closely related to S. kraussei and S. silvaticum.

Production of enriched in B vitamins biomass of Yarrowia lipolytica grown in biofuel waste.

Yarrowia lipolytica as an oleaginous yeast is capable of growing in various non-conventional hydrophobic substrate types, especially industrial wastes. In this study, the content of thiamine (vitamin B1), riboflavin (vitamin B2), pyridoxine (vitamin B6), biotin (vitamin B7) and folic acid (vitamin B9) in the wet biomass of Y. lipolytica strains cultivated in biofuel waste (SK medium), compared to the standard laboratory YPD medium, was assessed. Additionally, the biomass of Y. lipolytica A-101 grown in biofuel waste (SK medium) was dried and examined for B vitamins concentration according to the recommended microbial methods by AOAC Official Methods. The mean values of these vitamins per 100 g of dry weight of Y. lipolytica grown in biofuel waste (SK medium) were as follows: thiamine 1.3 mg/100 g, riboflavin 5.3 mg/100 g, pyridoxine 4.9 mg/100 g, biotin 20.0 µg/100 g, and folic acid 249 µg/100 g. We have demonstrated that the dried biomass is a good source of B vitamins which can be used as nutraceuticals to supplement human diet, especially for people at risk of B vitamin deficiencies in developed countries. Moreover, the biodegradation of biofuel waste by Y. lipolytica is desired for environmental protection.

Nanopore-Sequencing Characterization of the Gut Microbiota of Melolontha melolontha Larvae: Contribution to Protection against Entomopathogenic Nematodes?

This study focused on the potential relationships between midgut microbiota of the common cockchafer Melolontha melolontha larvae and their resistance to entomopathogenic nematodes (EPN) infection. We investigated the bacterial community associated with control and unsusceptible EPN-exposed insects through nanopore sequencing of the 16S rRNA gene. Firmicutes, Proteobacteria, Actinobacteria, and Bacteroidetes were the most abundant bacterial phyla within the complex and variable midgut microbiota of the wild M. melolontha larvae. The core microbiota was found to include 82 genera, which accounted for 3.4% of the total number of identified genera. The EPN-resistant larvae differed significantly from the control ones in the abundance of many genera belonging to the Actinomycetales, Rhizobiales, and Clostridiales orders. Additionally, the analysis of the microbiome networks revealed different sets of keystone midgut bacterial genera between these two groups of insects, indicating differences in the mutual interactions between bacteria. Finally, we detected Xenorhabdus and Photorhabdus as gut residents and various bacterial species exhibiting antagonistic activity against these entomopathogens. This study paves the way to further research aimed at unravelling the role of the host gut microbiota on the output of EPN infection, which may contribute to enhancement of the efficiency of nematodes used in eco-friendly pest management.

Bacteria from the Midgut of Common Cockchafer (Melolontha melolontha L.) Larvae Exhibiting Antagonistic Activity Against Bacterial Symbionts of Entomopathogenic Nematodes: Isolation and Molecular Identification.

The mechanisms of action of the complex including entomopathogenic nematodes of the genera Steinernema and Heterorhabditis and their mutualistic partners, i.e., bacteria Xenorhabdus and Photorhabdus, have been well explained, and the nematodes have been commercialized as biological control agents against many soil insect pests. However, little is known regarding the nature of the relationships between these bacteria and the gut microbiota of infected insects. In the present study, 900 bacterial isolates that were obtained from the midgut samples of Melolontha melolontha larvae were screened for their antagonistic activity against the selected species of the genera Xenorhabdus and Photorhabdus. Twelve strains exhibited significant antibacterial activity in the applied tests. They were identified based on 16S rRNA and rpoB, rpoD, or recA gene sequences as Pseudomonas chlororaphis, Citrobacter murliniae, Acinetobacter calcoaceticus, Chryseobacterium lathyri, Chryseobacterium sp., Serratia liquefaciens, and Serratia sp. The culture filtrate of the isolate P. chlororaphis MMC3 L3 04 exerted the strongest inhibitory effect on the tested bacteria. The results of the preliminary study that are presented here, which focused on interactions between the insect gut microbiota and mutualistic bacteria of entomopathogenic nematodes, show that bacteria inhabiting the gut of insects might play a key role in insect resistance to entomopathogenic nematode pressure.

Steinernema poinari (Nematoda: Steinernematidae): a new symbiotic host of entomopathogenic bacteria Xenorhabdus bovienii.

Three strains of symbiotic bacteria were isolated from an entomopathogenic nematode Steinernema poinari retrieved from soil in eastern Poland. Using 16S rDNA, recA, gltX, gyrB, and dnaN gene sequences for phylogenetic analysis, these strains were shown to belong to the species Xenorhabdus bovienii. The nucleotide identity between the studied S. poinari microsymbionts and other X. bovienii strains calculated for 16S rDNA and concatenated sequences of four protein-coding genes was 98.7-100% and 97.9-99.5%, respectively. The phenotypic properties of the isolates also supported their close phylogenetic relationship with X. bovienii. All three tested X. bovienii strains of different Steinernema clade origin supported the recovery of infective juveniles and subsequent development of the nematode population. However, the colonization degree of new infective juvenile generations was significantly affected by the bacterial host donor/recipient. The colonization degree of infective juveniles reared on bacterial symbionts deriving from a non-cognate clade of nematodes was extremely low, but proved the possible host-switching between non-related Steinernema species.

Strains of Photorhabdus spp. associated with polish Heterorhabditis isolates: their molecular and phenotypic characterization and symbiont exchange.

The relationships between six bacterial symbionts of the entomopathogenic nematodes Heterorhabditis bacteriophora and Heterorhabditis megidis from Poland to species and subspecies of the genus Photorhabdus were evaluated. This study was based on phylogenetic analysis of sequence data of five genes: 16S rRNA, gyrB, recA, gltX, and dnaN. The bacteria were also characterized phenotypically by biochemical and physiological tests. Our results have revealed that the Photorhabdus strains isolated from H. megidis belong to P. temperata, subsp. temperata and subsp. cinerea. Isolates from H. bacteriophora represent P. luminescens subs. kayaii and P. temperata subs. cinerea. This study for the first time provides evidence for H. bacteriophora and P. temperata subsp. cinerea symbiotic association. In addition, we tested whether the microsymbionts of the Polish H. bacteriophora and H. megidis isolates support the development of non-native nematode host population and colonization of their infective juveniles. It has been shown that the studied Photorhabdus strains can readily swap their nematode host, both at intra- and interspecies level. It supports the hypothesis of different symbiotic associations in the Heterorhabditis-Photorhabdus lineage.

Molecular and phenotypic characterization of Xenorhabdus bovienii symbiotically associated with Steinernema silvaticum.

Steinernema silvaticum is a common entomopathogenic nematode in soil of Europe; however, little is known about the bacteria living in symbiosis with this animal. In this study, we have isolated four bacterial strains from S. silvaticum and identified them as members of the species Xenorhabdus bovienii. This study was based on 16S rRNA and concatenated recA, dnaN, gltX, and gyrB gene sequence analysis. In addition, phenotypic traits have been considered, indicating that the tested strains are the most similar to those of X. bovienii. The phylogenetic relationships between the isolated strains and other strains of X. bovienii derived from various nematode hosts were analyzed and discussed. This is the first report confirming the symbiotic association of X. bovienii with S. silvaticum.

Catalytic activity of mutants of yeast protein kinase CK2alpha.

Yeast CK2 is a highly conserved member of the protein kinase CGMC subfamily composed of two catalytic (alpha and alpha') and two regulatory (beta and beta') subunits. The amino-acid sequences of both catalytic subunits are only 60% homologous. Modelling of the tertiary structure of the CK2alpha displays additional alpha-helical structures not present in the CK2alpha' subunit, connecting the ATP-binding loop with the catalytic and activation loops. Deletion of this part causes drastic structural and enzymatic changes of the protein (CK2alpha(Delta91-128)) with characteristics similar to yeast CK2alpha' (low sensitivity to salt, heparin and spermine). Additionally, the deletion causes an over 5-fold decrease of the binding affinity for ATP and ATP-competitive inhibitors (TBBt and TBBz). The structural basis for TBBt and TBBz selectivity is provided by the hydrophobic pocket adjacent to the ATP/GTP binding site, which is smaller in CK2 than in the majority of other protein kinases. The importance of hydrophobic interactions in the binding of specific inhibitors was investigated here by mutational analysis of CK2alpha residues whose side chains contribute to reducing the size of the hydrophobic pocket. Site-directed mutagenesis was used to replace Val67 and Ile213 by Ala. The kinetic properties of the single mutants CK2alpha(Val67Ala) and CK2alpha(Ile213Ala), and the double mutant CK2(Val67Ala Ile213Ala) were studied with respect to ATP, and both inhibitors TBBt and TBBz. The K(m) values for ATP did not change or were very close to those of the parental kinase. In contrast, all CK2alpha mutants analysed displayed higher K(i) values towards the inhibitors (10 to 12-fold higher with TBBt and 3 to 6-fold with TBBt) comparing to recombinant wild-type CK2alpha.

Yeast holoenzyme of protein kinase CK2 requires both beta and beta' regulatory subunits for its activity.

Protein kinase CK2 is a highly conserved Ser/Thr protein kinase that is ubiquitous among eucaryotic organisms and appears to play an important role in many cellular functions. This enzyme in yeast has a tetrameric structure composed of two catalytic (alpha and/or alpha') subunits and two regulatory beta and beta' subunits. Previously, we have reported isolation from yeast cells four active forms of CK2, composed of alphaalpha'betabeta', alpha2betabeta', alpha'2betabeta' and a free alpha'-catalytic subunit. Now, we report that in Saccharomyces cerevisiae CK2 holoenzyme regulatory beta subunit cannot substitute other beta' subunit and only both of them can form fully active enzymatic unit. We have examined the subunit composition of tetrameric complexes of yeast CK2 by transformation of yeast strains containing single deletion of the beta or beta' regulatory subunits with vectors carrying lacking CKB1 or CKB2 genes. CK2 holoenzyme activity was restored only in cases when both of them were present in the cell. Additional, co-immunoprecypitation experiments show that polyadenylation factor Fip1 interacts with catalytic alpha subunits of CK2 and interaction with beta subunits in the holoenzyme decreases CK2 activity towards this protein substrate. These data may help to elucidate the role of yeast protein kinase CK2beta/beta' subunits in the regulation of holoenzyme assembly and phosphotransferase activity.

Different properties of four molecular forms of protein kinase CK2 from Saccharomyces cerevisiae.

CK2 is a pleiotropic constitutively active serine/threonine protein kinase composed of two catalytic alpha- and two regulatory beta-subunits, whose regulation is still not well understood. It seems to play an essential role in regulation of many cellular processes. Four active forms of CK2, composed of alphaalpha'betabeta', alpha(2)betabeta', alpha'(2)betabeta', and a free alpha'-subunit were isolated from wild-type yeast and strains containing a single deletion of the catalytic subunit. Each species exhibits properties typical for CK2, but they differ in substrate specificity and sensitivity to inhibitors. This suggests that each CK2 isomer may regulate different process or may differ in the way of its regulation.

Characterization of phages virulent for Sarothamnus scoparius bradyrhizobia.

Four virulent phages--PhiDl, PhiTl, PhiCYT21, and PhiOS6, infective on Sarothamnus scoparius rhizobia--were isolated from the soil and characterized for morphology, host range, rate of adsorption to bacterial cells, and genome size. New phages were separated into two morphological families: Siphoviridae with long, noncontractile tails (PhiDl, PhiTl) and Myoviridae with long, contractile tails (PhiCYT21, PhiOS6). They were also classified into two groups by a host specificity. One of them included viruses (PhiDl and PhiTl) that lysed S. scoparius bradyrhizobia and Bradyrhizobium sp. (Lupinus) strain Dl, and the second one comprised phages (PhiCYT21 and PhiOS6) that parasitized only Scotch broom native microsymbionts. Phages specific for S. scoparius rhizobia were differentiated not only by morphology and host range but also by a genome size that was in the range from 47,583 to 60,098 b.p.