Comparative functional genomic analysis of two <i>Vibrio </i>phages reveals complex metabolic interactions with the host cell

Sequencing and annotation was performed for two giant double stranded DNA bacteriophages, φGrn1 and φSt2 of the Myoviridae family, considered to be of great interest for phage therapy against Vibrios in aquaculture live feeds. In addition, phage-host metabolic interactions and exploitation was studied by transcript profiling of selected viral and host genes. Comparative genomic analysis with other giant Vibrio phages was also performed to establish the presence and location of homing endonucleases highlighting distinct features for both phages. Phylogenetic analysis revealed that they belong to the schizoT4like clade. Although many reports of newly sequenced viruses have provided a large set of information, basic research related to the shift of the bacterial metabolism during infection remains stagnant. The function of many viral protein products in the process of infection is still unknown. Genome annotation identified the presence of several viral ORFs participating in metabolism, including a Sir2/cobB (sirtuin) protein and a number of genes involved in auxiliary NAD+ and nucleotide biosynthesis, necessary for phage DNA replication. Key genes were subsequently selected for detail study of their expression levels during infection. This work suggests a complex metabolic interaction and exploitation of the host metabolic pathways and biochemical processes, including a possible post-translational protein modification, by the virus during infection

Morphological, Physiological and Metabolomic Response of transgenic tobacco plants (N. tabacum L.) overexpressing GmGSTU4 under Drought Stress

GSTs appear to have a significant role in plants’ adaptation under abiotic stress as many isoenzymes are found to be differentially expressed under these conditions yet, little is known about the regulatory functions of GSTs. Wild type and transgenic tobacco plants over-expressing the soybean GmGSTU4 of cultivars Basmas, Burley and Virginia were grown in vitro under 100 and 200mM mannitol or in soil (plant pots) by withholding watering for 15 days. However, GmGSTU4 plants did not exhibit significant differences in drought tolerance compared to wild-type plants. Morphological (shoot length, total and root fresh weight) and physiological (chlorophyll content, relative water content and photosynthetic capacity) parameters of transgenic plants did not differ from the wild-type in the presence of 100 or 200mM mannitol or in the soil when watering was halted. Metabolite profiling was used to understand the dynamics between the wild-type and transgenic tobacco response to drought stress. Different metabolic pathways are involved in production of osmoprotectants. These molecules accumulate in plants under stress conditions as adaptive mechanism, which can provide stress tolerance. GmGSTU4 plants did not exhibit difference in drought tolerance compared to wild-type plants, however metabolomics analysis indicated alterations in metabolite profile and increased concentration of sorbitol, glycerol and pyruvic acid. In conclusion, overexpression of GmGSTU4 in transgenic plants did not affect their drought stress tolerance although it has altered their metabolite profile possible because of diverse effects on plant stress tolerance mechanism

Maintenance of metabolic homeostasis and induction of cytoprotectants and secondary metabolites in alachlor-treated GmGSTU4-overexpressing tobacco plants, as resolved by metabolomics

Herbicides are an invaluable tool for agricultural production scaling up. However, their continuous and intensive use has led to an increased incidence of herbicide resistant weeds and environmental pollution. Plant glutathione transferases (GSTs) are tightly connected with crop and weed herbicide tolerance capacitating their efficient metabolic detoxification, thus GSTs can be biotechnologically exploited towards addressing those issues. However, information on their effects at a “systems” level in response to herbicides is lacking. Here, we aimed to study the effects of the chloroacetanilide herbicide alachlor on the metabolome of wild-type and tobacco plants overexpressing the soybean tau class glutathione transferase GmGSTU4. Alachlor-treated wild-type plants This system, naturally serving the detoxification of endogenous exhibited an abiotic stress-like response with increased abundance of compatible solutes, decrease in TCA cycle intermediates and decreased sugar and amino acid content. Transgenic plants responded distinctly, exhibiting an increased induction of abiotic stress responsive metabolites, accumulation of secondary metabolites and its precursors, and metabolic detoxification by-products compared to wild-type plants. These results suggest that the increased metabolic capacity of GmGSTU4 overexpressing plants is accompanied by pleiotropic metabolic alterations, which could be the target for further manipulation in order to develop herbicide resistant crops, plants with increased phytoremediation potential, as well as efficient management of non-target site, GST induced, herbicide resistance in weeds

A cytosolic invertase is required for normal growth and cell development in the model legume, Lotus japonicus

Neutral/alkaline invertases are a subgroup, confined to plants and cyanobacteria, of a diverse family of enzymes. A family of seven closely-related genes, LjINV1–LjINV7, is described here and their expression in the model legume, Lotus japonicus, is examined. LjINV1 previously identified as encoding a nodule-enhanced isoform is the predominant isoform present in all parts of the plant. Mutants for two isoforms, LjINV1 and LjINV2, were isolated using TILLING. A premature stop codon allele of LjINV2 had no effect on enzyme activity nor did it show a visible phenotype. For LjINV1, premature stop codon and missense mutations were obtained and the phenotype of the mutants examined. Recovery of homozygous mutants was problematic, but their phenotype showed a severe reduction in growth of the root and the shoot, a change in cellular development, and impaired flowering. The cellular organization of both roots and leaves was altered; leaves were smaller and thicker with extra layers of cells and roots showed an extended and broader zone of cell division. Moreover, anthers contained no pollen. Both heterozygotes and homozygous mutants showed decreased amounts of enzyme activity in nodules and shoot tips. Shoot tips also contained up to a 9-fold increased level of sucrose. However, mutants were capable of forming functional root nodules. LjINV1 is therefore crucial to whole plant development, but is clearly not essential for nodule formation or function

Changes in the Rumen Bacteriome Structure and Enzymatic Activities of Goats in Response to Dietary Supplementation with Schizochytrium spp.

With the aim to produce functional dairy products enriched with polyunsaturated fatty acids (PUFA) by using feed supplements, radical changes could occur in the rumen microbiome. This work investigated the alterations of the rumen bacteriome of goats fed with PUFA-rich marine microalgae Schizochytrium spp. For the trial, twenty-four goats were divided into four homogenous clusters (six goats/treatment) according to their fat-corrected (4%) milk yield, body weight, and age; they were individually fed with alfalfa hay and a concentrate (F/C = 50/50). The concentrate of the control group (CON) contained no microalgae, while those of the treated groups were supplemented daily with 20 (ALG20), 40 (ALG40), and 60 g (ALG60) of Schizochytrium spp./goat. Rumen fluid samples were collected using a stomach tube during the 20th and 40th days of the experiment. The microbiome analysis using a 16S rRNA sequencing platform revealed that Firmicutes were decreased in microalgae-fed goats, while Bacteroidetes showed a tendency to increase in the ALG40 group due to the enhancement of Prevotellaceae. Cellulolytic bacteria, namely Treponema bryantii, Ruminococcus gauvreauii, R. albus, and R. flavefaciens, were decreased in the ALG40 group, resulting in an overall decrease of cellulase activity. In contrast, the amylolytic potential was significantly enhanced due to an upsurge in Ruminobacter amylophilus, Succinivibrio dextrinosolvens, and Fretibacterium fastidiosum populations. In conclusion, supplementing goats’ diets with 20 g Schizochytrium spp. could be considered a sustainable and efficient nutritional strategy to modulate rumen microbiome towards the development of dairy products enriched with bioactive compounds, while higher levels induced substantial shifts in determinant microbes’ populations

Feeding level regulates the expression of some genes involved with programed cell death and remodeling in goat and sheep mammary tissue

Mammary tissue (MT) turnover is characterized by programed cell death and remodeling which might be affected by both feeding level and animal species. Thus, twenty-four dairy goats and the same number of sheep were assigned to three homogenous sub-groups per animal species and fed the same diet in quantities which met 70% (FL70), 100% (FL100) and 130% (FL130) of their daily energy and crude protein requirements. Individual MT samples were taken by biopsy from the animals on the 30th and 60th experimental day. The results showed, in the first sampling time, a significant reduction in the mRNA abundance for selected genes involved in programed cell death in both FL 70 fed goats (STAT3 and BECN1) and sheep (CASPASE8 and BECN1) compared with the respective FL100 groups. The FL130, in comparison with the FL100, caused a significant increase in transcripts accumulation of STAT3 gene in both sampling times and CASPASE8 gene in the second sampling time in goat MT, while the opposite happened for the mRNA expression of CASPASE8 and BECN1 genes in sheep MT, but only in the first sampling time. Moreover, a significant up regulation in the mRNA levels of MMP2 gene in MT of FL130 fed sheep was observed. The FL130, in comparison with the FL70, caused an enhancement in the mRNA expression levels of BECN1, CASPASE8, BAX and STAT3 genes in goat MT only. It was also shown that apoptosis and autophagy can be affected simultaneously by the feeding level. Overfeeding affects MT programed cell death and remodeling by a completely different way in goats than sheep. In conclusion, feeding level and animal species have strong effects on both MT programed cell death (apoptosis and autophagy) and remodeling but the molecular mechanisms need further investigation

The Effect of Forage-to-Concentrate Ratio on Schizochytrium spp.-Supplemented Goats: Modifying Rumen Microbiota

The inclusion of feed additives and the implementation of various nutritional strategies are studied to modify the rumen microbiome and consequently its function. Nevertheless, rumen enzymatic activity and its intermediate products are not always matched with the microbiome structure. To further elucidate such differences a two-phase trial using twenty-two dairy goats was carried out. During the first phase, both groups (20HF n = 11; high forage and 20HG n = 11; high grain) were supplemented with 20 g Schizochytrium spp./goat/day. The 20HF group consumed a diet with a forage:concentrate (F:C) ratio of 60:40 and the 20HG-diet consisted of a F:C = 40:60. In the second phase, the supplementation level of Schizochytrium spp. was increased to 40 g/day/goat while the F:C ratio between the two groups were remained identical (40HF n = 11; high forage and 40HG n = 11; high grain). By utilizing a next-generation sequencing technology, we monitored that the high microalgae inclusion level and foremost in combination with a high grains diet increased the unmapped bacteria within the rumen. Bacteroidetes and Prevotella brevis were increased in the 40HG -fed goats as observed by using a qPCR platform. Additionally, methanogens and Methanomassiliicoccales were increased in high microalgae-fed goats, while Methanobrevibacter and Methanobacteriales were decreased. Fibrolytic bacteria were decreased in high microalgae-fed goats, while cellulolytic activity was increased. Ammonia was decreased in high grains-fed goats, while docosapentaenoic and docosahexaenoic acids showed a lower degradation rate in the rumen of high forage-fed goats. The alteration of the F:C ratio in goats supplemented with Schizochytrium spp. levels modified both ruminal microbiota and enzymatic activity. However, there was no significant consistency in the relations between them

The Impact of Whole Sesame Seeds on the Expression of Key-Genes Involved in the Innate Immunity of Dairy Goats

Whole sesame seeds (WSS) are rich in both linoleic acid (LA) and lignans. However, their impact on the innate immunity of goats is not well studied. Twenty-four goats were divided into three homogeneous sub-groups; comprise one control (CON) and two treated (WWS5 and WWS10). In the treated groups, WSS were incorporated in the concentrates of the CON at 5 (WSS5) and 10% (WSS10) respectively, by partial substitution of both soybean meal and corn grain. The expression levels of MAPK1, IL6, TRIF, IFNG, TRAF3, and JUND genes in the neutrophils of WSS10 fed goats were reduced significantly compared with the CON. The same was found for the expression levels of IFNG and TRAF3 genes in the neutrophils of WSS5 fed goats. Both treated groups primarily affected the MYD88-independent pathway. The dietary supplementation of goats with WSS might be a good nutritional strategy to improve their innate immunity

The impact of rumen-protected amino acids on the expression of key- genes involved in the innate immunity of dairy sheep.

Rumen protected amino acids inclusion in ewes' diets has been proposed to enhance their innate immunity. The objective of this work was to determine the impact of dietary supplementation with rumen-protected methionine or lysine, as well as with a combination of these amino acids in two different ratios, on the expression of selected key-genes (NLRs, MyD88, TRIF, MAPK-1, IRF-3, JunD, TRAF-3, IRF-5, IL-1α, IL-10, IKK-α, STAT-3 and HO-1). Thus, sixty Chios dairy ewes (Ovis aries) were assigned to one of the following five dietary treatments (12 animals/ treatment): A: basal diet consist of concentrates, wheat straw and alfalfa hay (control group); B: basal diet +6.0 g/head rumen-protected methionine; C: basal diet + 5.0 g/head rumen-protected lysine; D: basal diet +6.0 g/head rumen-protected methionine + 5.0 g/head rumen-protected lysine and E: basal diet +12.0 g/head rumen-protected methionine + 5.0 g/head rumen-protected lysine. The results revealed a significant downregulation of relative transcript level of the IL-1α gene in the neutrophils of C and in monocytes of D ewes compared with the control. Significantly lower mRNA transcript accumulation was also observed for the MyD88 gene in the neutrophils of ewes fed with lysine only (C). The mRNA relative expression levels of JunD gene were highly induced in the monocytes, while those of IL-10 and HO-1 genes were declined in the neutrophils of ewes fed with the C and D diets compared with the control. Lower transcript levels of STAT-3 gene were observed in the neutrophils of ewes fed with either C or with E diets in comparison with the control. In conclusion, our results suggest that the dietary supplementation of ewes with rumen-protected amino acids, down regulate the expression of some genes involved in the pro-inflammatory signalling