Nasal carriage of Staphylococcus aureus in healthy dairy cows in Algeria: antibiotic resistance, enterotoxin genes and biofilm formation.

BACKGROUND: Staphylococcus aureus can colonize and infect a variety of animal species. In dairy herds, it is one of the leading causes of mastitis cases. The objective of this study was to characterize the S. aureus isolates recovered from nasal swabs of 249 healthy cows and 21 breeders of 21 dairy farms located in two provinces of Algeria (Tizi Ouzou and Bouira). METHODS: The detection of enterotoxin genes was investigated by multiplex PCRs. Resistance of recovered isolates to 8 antimicrobial agents was determined by disc-diffusion method. The slime production and biofilm formation of S. aureus isolates were assessed using congo-red agar (CRA) and microtiter-plate assay. Molecular characterization of selected isolates was carried out by spa-typing and Multi-Locus-Sequence-Typing (MLST). RESULTS: S. aureus was detected in 30/249 (12%) and 6/13 (28.6%) of nasal swabs in cows and breeders, respectively, and a total of 72 isolates were recovered from positive samples (59 isolates from cows and 13 from breeders). Twenty-six of these isolates (36.1%) harbored genes encoding for staphylococcal enterotoxins, including 17/59 (28.8%) isolates from cows and 9/13 (69.2%) from breeders. Moreover, 49.1% and 92.3% of isolates from cows and breeders, respectively, showed penicillin resistance. All isolates were considered as methicillin-susceptible (MSSA). Forty-five (76.3%) of the isolates from cows were slime producers and 52 (88.1%) of them had the ability to form biofilm in microtiter plates. Evidence of a possible zoonotic transmission was observed in two farms, since S. aureus isolates recovered in these farms from cows and breeders belonged to the same clonal lineage (CC15-ST15-t084 or CC30-ST34-t2228). CONCLUSIONS: Although healthy cows in this study did not harbor methicillin-resistant S. aureus isolates, the nares of healthy cows could be a reservoir of enterotoxigenic and biofilm producing isolates which could have implications in human and animal health.

Variation in Expression of the HECT E3 Ligase UPL3 Modulates LEC2 Levels, Seed Size, and Crop Yields in Brassica napus.

Identifying genetic variation that increases crop yields is a primary objective in plant breeding. We used association analyses of oilseed rape/canola (Brassica napus) accessions to identify genetic variation that influences seed size, lipid content, and final crop yield. Variation in the promoter region of the HECT E3 ligase gene BnaUPL3 C03 made a major contribution to variation in seed weight per pod, with accessions exhibiting high seed weight per pod having lower levels of BnaUPL3 C03 expression. We defined a mechanism in which UPL3 mediated the proteasomal degradation of LEC2, a master transcriptional regulator of seed maturation. Accessions with reduced UPL3 expression had increased LEC2 protein levels, larger seeds, and prolonged expression of lipid biosynthetic genes during seed maturation. Natural variation in BnaUPL3 C03 expression appears not to have been exploited in current B napus breeding lines and could therefore be used as a new approach to maximize future yields in this important oil crop.

Metabolic reconstructions identify plant 3-methylglutaconyl-CoA hydratase that is crucial for branched-chain amino acid catabolism in mitochondria.

The proteinogenic branched-chain amino acids (BCAAs) leucine, isoleucine and valine are essential nutrients for mammals. In plants, BCAAs double as alternative energy sources when carbohydrates become limiting, the catabolism of BCAAs providing electrons to the respiratory chain and intermediates to the tricarboxylic acid cycle. Yet, the actual architecture of the degradation pathways of BCAAs is not well understood. In this study, gene network modeling in Arabidopsis and rice, and plant-prokaryote comparative genomics detected candidates for 3-methylglutaconyl-CoA hydratase (4.2.1.18), one of the missing plant enzymes of leucine catabolism. Alignments of these protein candidates sampled from various spermatophytes revealed non-homologous N-terminal extensions that are lacking in their bacterial counterparts, and green fluorescent protein-fusion experiments demonstrated that the Arabidopsis protein, product of gene At4g16800, is targeted to mitochondria. Recombinant At4g16800 catalyzed the dehydration of 3-hydroxymethylglutaryl-CoA into 3-methylglutaconyl-CoA, and displayed kinetic features similar to those of its prokaryotic homolog. When at4g16800 knockout plants were subjected to dark-induced carbon starvation, their rosette leaves displayed accelerated senescence as compared with control plants, and this phenotype was paralleled by a marked increase in the accumulation of free and total leucine, isoleucine and valine. The seeds of the at4g16800 mutant showed a similar accumulation of free BCAAs. These data suggest that 3-methylglutaconyl-CoA hydratase is not solely involved in the degradation of leucine, but is also a significant contributor to that of isoleucine and valine. Furthermore, evidence is shown that unlike the situation observed in Trypanosomatidae, leucine catabolism does not contribute to the formation of the terpenoid precursor mevalonate.

A Dedicated Type II NADPH Dehydrogenase Performs the Penultimate Step in the Biosynthesis of Vitamin K1 in Synechocystis and Arabidopsis.

Mutation of Arabidopsis thaliana NAD(P)H DEHYDROGENASE C1 (NDC1; At5g08740) results in the accumulation of demethylphylloquinone, a late biosynthetic intermediate of vitamin K1. Gene coexpression and phylogenomics analyses showed that conserved functional associations occur between vitamin K biosynthesis and NDC1 homologs throughout the prokaryotic and eukaryotic lineages. Deletion of Synechocystis ndbB, which encodes for one such homolog, resulted in the same defects as those observed in the cyanobacterial demethylnaphthoquinone methyltransferase knockout. Chemical modeling and assay of purified demethylnaphthoquinone methyltransferase demonstrated that, by virtue of the strong electrophilic nature of S-adenosyl-l-methionine, the transmethylation of the demethylated precursor of vitamin K is strictly dependent on the reduced form of its naphthoquinone ring. NDC1 was shown to catalyze such a prerequisite reduction by using NADPH and demethylphylloquinone as substrates and flavine adenine dinucleotide as a cofactor. NDC1 displayed Michaelis-Menten kinetics and was markedly inhibited by dicumarol, a competitive inhibitor of naphthoquinone oxidoreductases. These data demonstrate that the reduction of the demethylnaphthoquinone ring represents an authentic step in the biosynthetic pathway of vitamin K, that this reaction is enzymatically driven, and that a selection pressure is operating to retain type II NAD(P)H dehydrogenases in this process.

Genetically Programmed Changes in Photosynthetic Cofactor Metabolism in Copper-deficient Chlamydomonas.

Genetic and genomic studies indicate that copper deficiency triggers changes in the expression of genes encoding key enzymes in various chloroplast-localized lipid/pigment biosynthetic pathways. Among these are CGL78 involved in chlorophyll biosynthesis and HPPD1, encoding 4-hydroxyphenylpyruvate dioxygenase catalyzing the committed step of plastoquinone and tocopherol biosyntheses. Copper deficiency in wild-type cells does not change the chlorophyll content, but a survey of chlorophyll protein accumulation in this situation revealed increased accumulation of LHCSR3, which is blocked at the level of mRNA accumulation when either CGL78 expression is reduced or in the crd1 mutant, which has a copper-nutrition conditional defect at the same step in chlorophyll biosynthesis. Again, like copper-deficient crd1 strains, cgl78 knock-down lines also have reduced chlorophyll content concomitant with loss of PSI-LHCI super-complexes and reduced abundance of a chlorophyll binding subunit of PSI, PSAK, which connects LHCI to PSI. For HPPD1, increased mRNA results in increased abundance of the corresponding protein in copper-deficient cells concomitant with CRR1-dependent increased accumulation of γ-tocopherols, but not plastoquinone-9 nor total tocopherols. In crr1 mutants, where increased HPPD1 expression is blocked, plastochromanol-8, derived from plastoquinone-9 and purported to also have an antioxidant function, is found instead. Although not previously found in algae, this metabolite may occur only in stress conditions.

The Origin and Biosynthesis of the Benzenoid Moiety of Ubiquinone (Coenzyme Q) in Arabidopsis.

It is not known how plants make the benzenoid ring of ubiquinone, a vital respiratory cofactor. Here, we demonstrate that Arabidopsis thaliana uses for that purpose two separate biosynthetic branches stemming from phenylalanine and tyrosine. Gene network modeling and characterization of T-DNA mutants indicated that acyl-activating enzyme encoded by At4g19010 contributes to the biosynthesis of ubiquinone specifically from phenylalanine. CoA ligase assays verified that At4g19010 prefers para-coumarate, ferulate, and caffeate as substrates. Feeding experiments demonstrated that the at4g19010 knockout cannot use para-coumarate for ubiquinone biosynthesis and that the supply of 4-hydroxybenzoate, the side-chain shortened version of para-coumarate, can bypass this blockage. Furthermore, a trans-cinnamate 4-hydroxylase mutant, which is impaired in the conversion of trans-cinnamate into para-coumarate, displayed similar defects in ubiquinone biosynthesis to that of the at4g19010 knockout. Green fluorescent protein fusion experiments demonstrated that At4g19010 occurs in peroxisomes, resulting in an elaborate biosynthetic architecture where phenylpropanoid intermediates have to be transported from the cytosol to peroxisomes and then to mitochondria where ubiquinone is assembled. Collectively, these results demonstrate that At4g19010 activates the propyl side chain of para-coumarate for its subsequent ß-oxidative shortening. Evidence is shown that the peroxisomal ABCD transporter (PXA1) plays a critical role in this branch.

Alterations in seed development gene expression affect size and oil content of Arabidopsis seeds.

Seed endosperm development in Arabidopsis (Arabidopsis thaliana) is under control of the polycomb group complex, which includes Fertilization Independent Endosperm (FIE). The polycomb group complex regulates downstream factors, e.g. Pheres1 (PHE1), by genomic imprinting. In heterozygous fie mutants, an endosperm develops in ovules carrying a maternal fie allele without fertilization, finally leading to abortion. Another endosperm development pathway depends on MINISEED3 (a WRKY10 transcription factor) and HAIKU2 (a leucine-rich repeat kinase). While the role of seed development genes in the embryo and endosperm establishment has been studied in detail, their impact on metabolism and oil accumulation remained unclear. Analysis of oil, protein, and sucrose accumulation in mutants and overexpression plants of the four seed development genes revealed that (1) seeds carrying a maternal fie allele accumulate low oil with an altered composition of triacylglycerol molecular species; (2) homozygous mutant seeds of phe1, mini3, and iku2, which are smaller, accumulate less oil and slightly less protein, and starch, which accumulates early during seed development, remains elevated in mutant seeds; (3) embryo-specific overexpression of FIE, PHE1, and MINI3 has no influence on seed size and weight, nor on oil, protein, or sucrose content; and (4) overexpression of IKU2 results in seeds with increased size and weight, and oil content of overexpressed IKU2 seeds is increased by 35%. Thus, IKU2 overexpression represents a novel strategy for the genetic manipulation of the oil content in seeds.

Phenotypic and Genotypic Characterization of Staphylococcus aureus Isolated from Nasal Samples of Healthy Dairy Goats in Algeria.

The present study aimed to determine the phenotypic and genotypic characteristics of S. aureus isolates from the nasal swabs of goats. A total of 232 nasal samples (one per animal) were collected from goats on 13 farms located in two regions of Algeria and were analyzed for the presence of S. aureus. The detection of virulence factors was carried out using PCR. The antibiotic susceptibility of the recovered isolates was assessed using the disc diffusion method. The biofilm formation ability was assessed by the Congo red agar method and a microtiter plate assay, and the molecular characterization of isolates was carried out by spa-typing, and for selected isolates also by multilocus sequence typing (MLST). Overall, 36 out of 232 nasal swabs (15.5%) contained S. aureus, and 62 isolates were recovered. Regarding the virulence factors, at least one staphylococcal enterotoxin gene was detected in 30 (48.4%) isolates. The gene tst encoding the toxic shock syndrome toxin was detected in fifteen isolates (24.2%), but none of the isolates harbored the gene of Panton-Valentine leukocidin (lukF/S-PV). Nine different spa-types were identified, including the detection of a new one (t21230). The recovered isolates were assigned to three clonal complexes, with CC5 (51.8%) being the most common lineage. Two isolates were methicillin-resistant (MRSA) and belonged to ST5 (CC5) and to spa-types t450 and t688. Moreover, 27 (43.5%) of the S. aureus isolates were found to be slime producers in Congo red agar, and all of the recovered isolates could produce biofilms in the microtiter plate assay. Our study showed that the nares of healthy goats could be a reservoir of toxigenic and antibiotic-resistant strains of S. aureus isolates, including MRSA, which could have implications for public health.

Acyl editing and headgroup exchange are the major mechanisms that direct polyunsaturated fatty acid flux into triacylglycerols.

Triacylglycerols (TAG) in seeds of Arabidopsis (Arabidopsis thaliana) and many plant species contain large amounts of polyunsaturated fatty acids (PUFA). These PUFA are synthesized on the membrane lipid phosphatidylcholine (PC). However, the exact mechanisms of how fatty acids enter PC and how they are removed from PC after being modified to participate in the TAG assembly are unclear, nor are the identities of the key enzymes/genes that control these fluxes known. By reverse genetics and metabolic labeling experiments, we demonstrate that two genes encoding the lysophosphatidylcholine acyltransferases LPCAT1 and LPCAT2 in Arabidopsis control the previously identified "acyl-editing" process, the main entry of fatty acids into PC. The lpcat1/lpcat2 mutant showed increased contents of very-long-chain fatty acids and decreased PUFA in TAG and the accumulation of small amounts of lysophosphatidylcholine in developing seeds revealed by [¹4C]acetate-labeling experiments. We also showed that mutations in LPCATs and the PC diacylglycerol cholinephosphotransferase in the reduced oleate desaturation1 (rod1)/lpcat1/lpcat2 mutant resulted in a drastic reduction of PUFA content in seed TAG, accumulating only one-third of the wild-type level. These results indicate that PC acyl editing and phosphocholine headgroup exchange between PC and diacylglycerols control the majority of acyl fluxes through PC to provide PUFA for TAG synthesis.

The Enterotoxin Gene Profiles and Enterotoxin Production of Staphylococcus aureus Strains Isolated from Artisanal Cheeses in Belgium.

A Staphyloccoccus aureus is one of the leading causes of food poisoning outbreaks (FPOs) worldwide. Staphylococcal food poisoning (SFP) is induced by the ingestion of food containing sufficient levels of staphylococcal enterotoxins (SEs). Currently, 33 SEs and SE-like toxins (SEls) have been described in the literature, but only five named "classical" enterotoxins are commonly investigated in FPOs due to lack of specific routine analytical techniques. The aims of this study were to (i) establish the genetic profile of strains in a variety of artisanal cheeses (n = 30) in Belgium, (ii) analyze the expression of the SE(l)s by these strains and (iii) compare the output derived from the different analytical tools. Forty-nine isolates of S. aureus were isolated from ten Belgian artisanal cheeses and were analyzed via microbiological, immunological, liquid chromatography mass spectrometry, molecular typing and genetic methods. The results indicated that classical SEs were not the dominant SEs in the Belgian artisanal cheeses that were analyzed in this study, and that all S. aureus isolates harbored at least one gene encoding a new SE(l). Among the new SE(l)s genes found, some of them code for enterotoxins with demonstrated emetic activity and ecg-enterotoxins. It is worth noting that the involvement of some of these new SEs has been demonstrated in SFP outbreaks. Thus, this study highlighted the importance of the development of specific techniques for the proper investigation of SFP outbreaks.

Seed-specific suppression of ADP-glucose pyrophosphorylase in Camelina sativa increases seed size and weight.

BACKGROUND: Camelina (Camelina sativa L.) is a promising oilseed crop that may provide sustainable feedstock for biofuel production. One of the major drawbacks of Camelina is its smaller seeds compared to other major oil crops such as canola, which limit oil yield and may also pose challenges in successful seedling establishment, especially in dryland cultivation. Previous studies indicate that seed development may be under metabolic control. In oilseeds, starch only accumulates temporarily during seed development but is almost absent in mature seeds. In this study, we explored the effect of altering seed carbohydrate metabolism on Camelina seed size through down-regulating ADP-glucose pyrophosphorylase (AGPase), a major enzyme in starch biosynthesis. RESULTS: An RNAi construct comprising sequences of the Camelina small subunit of an AGPase (CsAPS) was expressed in Camelina cultivar Suneson under a seed-specific promoter. The RNAi suppression reduced AGPase activities which concurred with moderately decreased starch accumulation during seed development. Transcripts of genes examined that are involved in storage products were not affected, but contents of sugars and water were increased in developing seeds. The transgenic seeds were larger than wild-type plants due to increased cell sizes in seed coat and embryos, and mature seeds contained similar oil but more protein contents. The larger seeds showed advantages on seedling emergence from deep soils. CONCLUSIONS: Changing starch and sugar metabolism during seed development may increase the size and mass of seeds without affecting their final oil content in Camelina. Increased seed size may improve seedling establishment in the field and increase seed yield.

Phylloquinone (Vitamin K1): Occurrence, Biosynthesis and Functions.

BACKGROUND: Phylloquinone is a prenylated naphthoquinone that is synthesized exclusively by plants, green algae, and some species of cyanobacteria, where it serves as a vital electron carrier in photosystem I and as an electron acceptor for the formation of protein disulfide bonds. OBJECTIVE: In humans and other vertebrates, phylloquinone plays the role of a vitamin (vitamin K1) that is required for blood coagulation and bone and vascular metabolism. Phylloquinone from green leafy vegetables and vegetable oil represents the major dietary source of vitamin K for humans. METHOD: In recent years, reverse genetics and biochemical approaches using the model plant Arabidopsis thaliana have shown that phylloquinone biosynthesis in plants involves paralogous and multifunctional enzymes, a compartmentation of the corresponding pathway in plastids and peroxisomes, and trafficking of some biosynthetic intermediates within plastids themselves. Furthermore, phylloquinone biosynthetic intermediates create crucial metabolic branch-points with other plastid-synthesized metabolites such as chlorophylls, tocopherols and salicylate. RESULTS & CONCLUSION: This review presents an update on recent studies of the central role of plastids in the biosynthesis of phylloquinone, in particular on the discovery of novel enzymatic steps that are likely paradigms for phylloquinone and menaquinone (vitamin K2)-synthesizing organisms alike.

Deciphering and modifying LAFL transcriptional regulatory network in seed for improving yield and quality of storage compounds.

Increasing yield and quality of seed storage compounds in a sustainable way is a key challenge for our societies. Genome-wide analyses conducted in both monocot and dicot angiosperms emphasized drastic transcriptional switches that occur during seed development. In Arabidopsis thaliana, a reference species, genetic and molecular analyses have demonstrated the key role of LAFL (LEC1, ABI3, FUS3, and LEC2) transcription factors (TFs), in controlling gene expression programs essential to accomplish seed maturation and the accumulation of storage compounds. Here, we summarize recent progress obtained in the characterization of these LAFL proteins, their regulation, partners and target genes. Moreover, we illustrate how these evolutionary conserved TFs can be used to engineer new crops with altered seed compositions and point out the current limitations. Last, we discuss about the interest of investigating further the environmental and epigenetic regulation of this network for the coming years.