Biodegradation of agave Comiteco bagasse by Pleurotus spp.: a source of cellulases useful in hydrolytic treatment to produce reducing sugars.

This study aimed to determine the production parameters of five strains of Pleurotus spp. during their cultivation on agave Comiteco bagasse, as well as the feasibility of using cellulolytic extracts to produce reducing sugars in the same bagasse. After cultivation, the basidiome production parameters varied between 41.2 and 65.7% (biological efficiency), 0.17 and 0.30 (yield), 0.60 and 0.90% (production rate), 16.4 and 41.1% (Bioconversion) and 9.4 and 21.3 g (mean mushroom weight). At day 15 of growth, P. djamor showed the highest ß-glucosidase activity (43.95 ± 4.5 IU/g); on day 33. The same strain had the highest endoglucanase activity (21.12 ± 0.5 IU/ml). Both extracts were partially purified, and the kinetic parameters Vmax and Km were estimated (20.83 µmole/ml sec and 232.01 µmole/ml for ß-glucosidase and 685.01 µmole/ml sec and 1,240.34 µmole/ml for endoglucanase). In the enzymatic hydrolysis assay, the highest concentration of reducing sugars (43.13 ± 1.09 g/L; 0.21 g/g bagasse) was obtained by a mixture of the two partially purified extracts acting synergistically after 48 h and with a pH adjustment. The results suggest that the use of agave Comiteco bagasse for cultivating edible mushrooms while obtaining cellulolytic extracts is an alternative treatment for waste reduction and valorization of agro-industrial by-products.

Nematocidal activity of hydroalcoholic extracts of spent substrate of Pleurotus djamor on L3 larvae of Haemonchus contortus.

The objective of this study was to evaluate and compare the in vitro lethal effect of the hydroalcoholic extract of the spent substrate of Pleurotus djamor ECS-123, obtained at 15 days of colonization (SPS) and at the first (SPS1) and second (SPS2) harvests, against infective larvae L3 of Haemonchus contortus. The in vitro lethal effect was evaluated by the L3 larval mortality test (LM) using six concentrations: 1.25, 2.5, 5, 10, 20, and 40 mg/mL, with ivermectin and thiabendazole (5 mg/mL) as controls. The first harvest extract (SPS1) of strain ECS-123 was subjected to liquid-liquid bipartition, which resulted in two fractions: aqueous (PdAcO) and ethyl acetate (PdAct). The chemical fractionation of PdAct with the highest mortality rate (80.11 %) was carried out with open-column chromatography, giving a total of 13 fractions, which were analyzed by thin-layer chromatography (TLC) and grouped into 5 mixtures (R1;1-3, R2;4-7, R3;8-9, R4;10-11 and R5;12-13). Subsequently, the mixtures were evaluated against H. contortus L3 larvae. Finally, the components of the mixtures with the highest nematocidal effects were evaluated by gas chromatography coupled to mass spectrometry (GC-MS). The data were analyzed with a completely randomized design through ANOVA using the generalized linear model (GLM) with the "R" program. The purification and characterization of R4 and R5 by GC-MS revealed the presence of the following compounds: veratryl alcohol, 4-hydroxy-3,5,5 trimethyl-4-[3-oxo-1-butenyl]-2- cyclohexen-1-one, caffeine and 5,6-dimethoxy-1(3 H) isobenzofuranone. This information allowed for the identification of nematocidal compounds in the degraded substrate of P. djamor, an activity that had not been reported previously.

Chemical Composition of an Anthelmintic Fraction of Pleurotus eryngii against Eggs and Infective Larvae (L3) of Haemonchus contortus.

This study was aimed at evaluating the in vitro effect of the edible mushroom (EM) Pleurotus eryngii against the eggs and larvae (L3) of Haemonchus contortus. The evaluation included acetone (AE) and hydroalcoholic (HA) extracts of the following strains: ECS-1138, ECS-1156, ECS-1255, ECS-1258, ECS-1261, ECS-1282, and ECS-1292. The HA extract of the ECS-1255 strain showed the highest effect on mortality rates of L3 (18.83%) at 20 µg/mL. After subjecting this HA extract to a normal phase chromatography column, five fractions were obtained; fraction F5 (100% MeOH) was the most effective against eggs, with hatching inhibition percentages of 88.77 and 91.87% at 20 and 40 mg/mL, respectively. Gas chromatography-mass spectrometry (GC-MS) subjected this fraction to an acetylation reaction to determine the content of the secondary metabolites. The GC-MS analysis showed that the F5 fraction was composed of trehalose CAS: 6138-23-4, polyols (L-iditol CAS: 488-45-9, galactitol CAS: 608-66-2, D-mannitol CAS: 69-65-8, D-glucitol CAS: 50-70-4, and myoinositol CAS: 87-89-8), adipic acid CAS: 124-04-9, stearic acid CAS: 57-11-4, squalene CAS: 111-02-4, and ß-sitosterol CAS: 83-46-5.

Genetic and Biochemical Analysis of Anaerobic Respiration in Bacteroides fragilis and Its Importance In Vivo.

In bacteria, the respiratory pathways that drive molecular transport and ATP synthesis include a variety of enzyme complexes that utilize different electron donors and acceptors. This property allows them to vary the efficiency of energy conservation and to generate different types of electrochemical gradients (H+ or Na+). We know little about the respiratory pathways in Bacteroides species, which are abundant in the human gut, and whether they have a simple or a branched pathway. Here, we combined genetics, enzyme activity measurements, and mammalian gut colonization assays to better understand the first committed step in respiration, the transfer of electrons from NADH to quinone. We found that a model gut Bacteroides species, Bacteroides fragilis, has all three types of putative NADH dehydrogenases that typically transfer electrons from the highly reducing molecule NADH to quinone. Analyses of NADH oxidation and quinone reduction in wild-type and deletion mutants showed that two of these enzymes, Na+-pumping NADH:quinone oxidoreductase (NQR) and NADH dehydrogenase II (NDH2), have NADH dehydrogenase activity, whereas H+-pumping NADH:ubiquinone oxidoreductase (NUO) does not. Under anaerobic conditions, NQR contributes more than 65% of the NADH:quinone oxidoreductase activity. When grown in rich medium, none of the single deletion mutants had a significant growth defect; however, the double Δnqr Δndh2 mutant, which lacked almost all NADH:quinone oxidoreductase activity, had a significantly increased doubling time. Despite unaltered in vitro growth, the single nqr deletion mutant was unable to competitively colonize the gnotobiotic mouse gut, confirming the importance of NQR to respiration in B. fragilis and the overall importance of respiration to this abundant gut symbiont.IMPORTANCEBacteroides species are abundant in the human intestine and provide numerous beneficial properties to their hosts. The ability of Bacteroides species to convert host and dietary glycans and polysaccharides to energy is paramount to their success in the human gut. We know a great deal about the molecules that these bacteria extract from the human gut but much less about how they convert those molecules into energy. Here, we show that B. fragilis has a complex respiratory pathway with two different enzymes that transfer electrons from NADH to quinone and a third enzyme complex that may use an electron donor other than NADH. Although fermentation has generally been believed to be the main mechanism of energy generation in Bacteroides, we found that a mutant lacking one of the NADH:quinone oxidoreductases was unable to compete with the wild type in the mammalian gut, revealing the importance of respiration to these abundant gut symbionts.

Occurrence of ferredoxin:NAD(+) oxidoreductase activity and its ion specificity in several Gram-positive and Gram-negative bacteria.

A ferredoxin:NAD(+) oxidoreductase was recently discovered as a redox-driven ion pump in the anaerobic, acetogenic bacterium Acetobacterium woodii. The enzyme is assumed to be encoded by the rnf genes. Since these genes are present in the genomes of many bacteria, we tested for ferredoxin:NAD(+) oxidoreductase activity in cytoplasmic membranes from several different Gram-positive and Gram-negative bacteria that have annotated rnf genes. We found this activity in Clostridium tetanomorphum, Clostridium ljungdahlii, Bacteroides fragilis, and Vibrio cholerae but not in Escherichia coli and Rhodobacter capsulatus. As in A. woodii, the activity was Na(+)-dependent in C. tetanomorphum and B. fragilis but Na(+)-independent in C. ljungdahlii and V. cholerae. We deleted the rnf genes from B. fragilis and demonstrated that the mutant has greatly reduced ferredoxin:NAD(+) oxidoreductase activity. This is the first genetic proof that the rnf genes indeed encode the reduced ferredoxin:NAD(+) oxidoreductase activity.

Inactivation of a single gene enables microaerobic growth of the obligate anaerobe Bacteroides fragilis.

Bacteroides fragilis can replicate in atmospheres containing ≤0.05% oxygen, but higher concentrations arrest growth by an unknown mechanism. Here we show that inactivation of a single gene, oxe (i.e., oxygen enabled) in B. fragilis allows for growth in concentrations as high as 2% oxygen while increasing the tolerance of this organism to room air. Known components of the oxidative stress response including the ahpC, kat, batA-E, and tpx genes were not individually important for microaerobic growth. However, a Δoxe strain scavenged H(2)O(2) at a faster rate than WT, indicating that reactive oxygen species may play a critical role in limiting growth of this organism to low-oxygen environments. Clinical isolates of B. fragilis displayed a greater capacity for growth under microaerobic conditions than fecal isolates, with some encoding polymorphisms in oxe. Additionally, isolation of oxygen-enabled mutants of Bacteroides thetaiotaomicron suggests that Oxe may mediate growth arrest of other anaerobes in oxygenated environments.

Sialic acid (N-acetyl neuraminic acid) utilization by Bacteroides fragilis requires a novel N-acetyl mannosamine epimerase.

We characterized the nanLET operon in Bacteroides fragilis, whose products are required for the utilization of the sialic acid N-acetyl neuraminic acid (NANA) as a carbon and energy source. The first gene of the operon is nanL, which codes for an aldolase that cleaves NANA into N-acetyl mannosamine (manNAc) and pyruvate. The next gene, nanE, codes for a manNAc/N-acetylglucosamine (NAG) epimerase, which, intriguingly, possesses more similarity to eukaryotic renin binding proteins than to other bacterial NanE epimerase proteins. Unphosphorylated manNAc is the substrate of NanE, while ATP is a cofactor in the epimerase reaction. The third gene of the operon is nanT, which shows similarity to the major transporter facilitator superfamily and is most likely to be a NANA transporter. Deletion of any of these genes eliminates the ability of B. fragilis to grow on NANA. Although B. fragilis does not normally grow with manNAc as the sole carbon source, we isolated a B. fragilis mutant strain that can grow on this substrate, likely due to a mutation in a NAG transporter; both manNAc transport and NAG transport are affected in this strain. Deletion of the nanE epimerase gene or the rokA hexokinase gene, whose product phosphorylates NAG, in the manNAc-enabled strain abolishes growth on manNAc. Thus, B. fragilis possesses a new pathway of NANA utilization, which we show is also found in other Bacteroides species.

Acetylornithine transcarbamylase: a novel enzyme in arginine biosynthesis.

Ornithine transcarbamylase is a highly conserved enzyme in arginine biosynthesis and the urea cycle. In Xanthomonas campestris, the protein annotated as ornithine transcarbamylase, and encoded by the argF gene, is unable to synthesize citrulline directly from ornithine. We cloned and overexpressed this X. campestris gene in Escherichia coli and show that it catalyzes the formation of N-acetyl-L-citrulline from N-acetyl-L-ornithine and carbamyl phosphate. We now designate this enzyme as an acetylornithine transcarbamylase. The K(m) values for N-acetylornithine and carbamyl phosphate were 1.05 mM and 0.01 mM, respectively. Additional putative transcarbamylases that might also be misannotated were found in the genomes of members of other xanthomonads, Cytophaga, and Bacteroidetes as well as in DNA sequences of bacteria from environmental isolates. It appears that these different paths for arginine biosynthesis arose very early in evolution and that the canonical ornithine transcarbamylase-dependent pathway became the prevalent form. A potent inhibitor, N(alpha)-acetyl-N(delta)-phosphonoacetyl-L-ornithine, was synthesized and showed a midpoint of inhibition at approximately 22 nM; this compound may prove to be a useful starting point for designing inhibitors specific to this novel family of transcarbamylases.

Cloning and expression of the human N-acetylglutamate synthase gene.

N-acetylglutamate synthase (NAGS, E.C. 2.3.1.1) is a mitochondrial enzyme catalyzing the formation of N-acetylglutamate (NAG), an essential allosteric activator of carbamylphosphate synthase I (CPSI), the first enzyme of the urea cycle. Patients with NAGS deficiency develop hyperammonemia because CPSI is inactive without NAG. The human NAGS cDNA was isolated from a liver library based on its similarity to mouse NAGS. The deduced amino acid sequence contains an N-terminal putative mitochondrial targeting signal of 49 amino acids (63% identity with mouse NAGS) followed by a "variable domain" of 45 amino acids (35% identity) and a "conserved domain" of 440 amino acids (92% identity). A cDNA sequence containing the "conserved domain" complements an NAGS-deficient Escherichia coli strain and the recombinant protein has arginine-responsive NAGS catalytic activity. The NAGS gene is expressed in the liver and small intestine; the intestinal transcript is smaller in size than liver transcript.

Crystal structure of a transcarbamylase-like protein from the anaerobic bacterium Bacteroides fragilis at 2.0 A resolution.

A transcarbamylase-like protein essential for arginine biosynthesis in the anaerobic bacterium Bacteroides fragilis has been purified and crystallized in space group P4(3)2(1)2 (a=b=153.4 A, c=94.8 A). The structure was solved using a single isomorphous replacement with anomalous scattering (SIRAS) and was refined at 2.0 A resolution to an R-factor of 20.6% (R-free=25.2%). The molecular model is trimeric and comprises 960 amino acid residues, two phosphate groups and 422 water molecules. The monomer has the consensus transcarbamylase fold with two structural domains linked by two long interdomain helices: the putative carbamoyl phosphate-binding domain and a binding domain for the second substrate. Each domain has a central parallel beta-sheet surrounded by alpha-helices and loops with alpha/beta topology. The putative carbamoyl phosphate-binding site is similar to those in ornithine transcarbamylases (OTCases) and aspartate transcarbamylases (ATCases); however, the second substrate-binding site is strikingly different. This site has several insertions and deletions, and residues critical to substrate binding and catalysis in other known transcarbamylases are not conserved. The three-dimensional structure and the fact that this protein is essential for arginine biosynthesis suggest strongly that it is a new member of the transcarbamylase family. A similar protein has been found in Xylella fastidiosa, a bacterium that infects grapes, citrus and other plants.

Identification, cloning and expression of the mouse N-acetylglutamate synthase gene.

In ureotelic animals, N-acetylglutamate (NAG) is an essential allosteric activator of carbamylphosphate synthetase I (CPSI), the first enzyme in the urea cycle. NAG synthase (NAGS; EC 2.3.1.1) catalyses the formation of NAG from glutamate and acetyl-CoA in liver and intestinal mitochondria. This enzyme is supposed to regulate ureagenesis by producing variable amounts of NAG, thus modulating CPSI activity. Moreover, inherited deficiencies in NAGS have been associated with hyperammonaemia, probably due to the loss of CPSI activity. Although the existence of the NAGS protein in mammals has been known for decades, the gene has remained elusive. We identified the mouse (Mus musculus) and human NAGS genes using their similarity to the respective Neurospora crassa gene. NAGS was cloned from a mouse liver cDNA library and was found to encode a 2.3 kb message, highly expressed in liver and small intestine with lower expression levels in kidney, spleen and testis. The deduced amino acid sequence contains a putative mitochondrial targeting signal at the N-terminus. The cDNA sequence complements an argA (NAGS)-deficient Escherichia coli strain, reversing its arginine auxotrophy. His-tagged versions of the pre-protein and two putative mature proteins were each overexpressed in E. coli, and purified to apparent homogeneity by using a nickel-affinity column. The pre-protein and the two putative mature proteins catalysed the NAGS reaction but one of the putative mature enzymes had significantly higher activity than the pre-protein. The addition of l-arginine increased the catalytic activity of the purified recombinant NAGS enzymes by approx. 2-6-fold.