Metagenomics for mining new genetic resources of microbial communities

Recent progress has revealed that the capture of genetic resources of complex microbial communities in metagenome libraries allows the discovery of a richness of new enzymatic diversity that had not previously been imagined. Activity-based screening of such libraries has demonstrated that this new diversity is not simply variations on known sequence themes, but rather the existence of entirely new sequence classes and novel functionalities. This new diversity, the surface of which has thus far only been scratched, constitutes potential for a wealth of new and improved applications in industry, medicine, agriculture, etc., and promises to facilitate in a significant manner our transition to a sustainable society, by contributing to the transition to renewable sources of energy, chemicals and materials, the lowering of pollutant burdens, lower processes energies, etc. Current bottlenecks in metagenomics include insufficient functional characterization and amplifying non-validated annotations of proteins in databases.     

Identification of RFLP and NBS/PK profiling markers for disease resistance loci in genetic maps of oats

Two of the domains most widely shared among R genes are the nucleotide binding site (NBS) and protein kinase (PK) domains. The present study describes and maps a number of new oat resistance gene analogues (RGAs) with two purposes in mind: (1) to identify genetic regions that contain R genes and (2) to determine whether RGAs can be used as molecular markers for qualitative loci and for QTLs affording resistance to Puccinia coronata. Such genes have been mapped in the diploid A. strigosa × A. wiestii (Asw map) and the hexaploid MN841801-1 × Noble-2 (MN map). Genomic and cDNA NBS-RGA probes from oat, barley and wheat were used to produce RFLPs and to obtain markers by motif-directed profiling based on the NBS (NBS profiling) and PK (PK profiling) domains. The efficiency of primers used in NBS/PK profiling to amplify RGA fragments was assessed by sequencing individual marker bands derived from genomic and cDNA fragments. The positions of 184 markers were identified in the Asw map, while those for 99 were identified in the MN map. Large numbers of NBS and PK profiling markers were found in clusters across different linkage groups, with the PK profiling markers more evenly distributed. The location of markers throughout the genetic maps and the composition of marker clusters indicate that NBS- and PK-based markers cover partly complementary regions of oat genomes. Markers of the different classes obtained were found associated with the two resistance loci, PcA and R-284B-2, mapped on Asw, and with five out of eight QTLs for partial resistance in the MN map. 53 RGA-RFLPs and 187 NBS/PK profiling markers were also mapped on the hexaploid map A. byzantina cv. Kanota × A. sativa cv. Ogle. Significant co-localization was seen between the RGA markers in the KO map and other markers closely linked to resistance loci, such as those for P. coronata and barley yellow dwarf virus (Bydv) that were previously mapped in other segregating populations.     

Effect of the Immobilization Method of Lipase from Thermomyces lanuginosus on Sucrose Acylation

Lipase from Thermomyces lanuginosus (formerly Humicola lanuginosa ) was immobilized using granulation by incubating low-particle-size silica with the lipase. Granules with a particle diameter in the range 0.3-1 &#117 mm were obtained. The immobilized lipase was tested in the acylation of sucrose with vinyl laurate in mixtures of tert -amyl alcohol: dimethyl sulfoxide. Results were compared with immobilization of enzyme by adsorption on polypropylene (Accurel EP100), deposition on Celite by precipitation, and covalent attachment to Eupergit C. Granulated lipase converted >95% of sucrose into 6- O -lauroylsucrose in 6 &#117 h. Accurel-lipase was also very active, converting 70% of sucrose into monoester in 2 &#117 h. The residual activity of granules after five reaction cycles under the best reaction conditions was 72%; this value was considerably higher than the one observed for the same lipase adsorbed on Accurel (15% residual activity after five cycles).     

CD studies of peptides that mimic the four helicoidal sequences of the uteroglobin monomer

Summary Short peptides spanning the helicoidal sequences of the uteroglobin monomer (crystal forms P2₁ and C222₁) were synthesized and studied by circular dichroism spectroscopy. None of them showed any secondary structure in the absence of HFIP. However, most peptides achieved a helical conformation when this structuring agent was used, with the exception of the analogue corresponding to the helicoidal fragment 19–24 (helix II, crystal P2₁). These results indicate that other factors, such as interchain interactions, have to contribute to helix stabilization in the molecule. On the other hand, while peptides corresponding to N- and C-terminal fragments that contain the first and fourth helices of the monomer, respectively (1–14 and 48–70) achieved a -like structure when 10–15% of HFIP was used, this behaviour was not observed when TFE was used. Moreover, substitution of cysteine by -aminobutyric acid at position 3 increased both the helicity of fragment 1–14 and its ability to adopt a -like structure, but the opposite effect was observed for fragment 48–70 when -aminobutyric acid was introduced at position 69. These results indicate that this part of the protein might be sensitive to the chemical environment it is exposed to and that the two cysteine residues at positions 3 and 69 of the monomer could play a different role in the folding process.     

Acetazolamide-M(II) [M(II) = Co(II),Ni(II),Cu(II)] complexes with ethylamine, diethylamine, triethylamine, and potassium hidroxide

Complexes of Co(II), Ni(II), and Cu(II) with dideprotonated Acm are synthesized and characterized. Acm acts as bidentate ligand through the N-sulfonamido atom and the N-thiadiazole atom except for K6CoAcm4.6H2O in which Acm behaves as monodentate through the N-sulfonamido atom.     

Disclosing Bias in Bisulfite Assay: MethPrimers Underestimate High DNA Methylation

Discordant results obtained in bisulfite assays using MethPrimers (PCR primers designed using MethPrimer software or assuming that non-CpGs cytosines are non methylated) versus primers insensitive to cytosine methylation lead us to hypothesize a technical bias. We therefore used the two kinds of primers to study different experimental models and methylation statuses. We demonstrated that MethPrimers negatively select hypermethylated DNA sequences in the PCR step of the bisulfite assay, resulting in CpG methylation underestimation and non-CpG methylation masking, failing to evidence differential methylation statuses. We also describe the characteristics of “Methylation-Insensitive Primers” (MIPs), having degenerated bases (G/A) to cope with the uncertain C/U conversion. As CpG and non-CpG DNA methylation patterns are largely variable depending on the species, developmental stage, tissue and cell type, a variable extent of the bias is expected. The more the methylome is methylated, the greater is the extent of the bias, with a prevalent effect of non-CpG methylation. These findings suggest a revision of several DNA methylation patterns so far documented and also point out the necessity of applying unbiased analyses to the increasing number of epigenomic studies.     

Three factors that modulate the activity of class D β-lactamases and interfere with the post-translational carboxylation of Lys70

The activity of class D β-lactamases is dependent on Lys70 carboxylation in the active site. Structural, kinetic and affinity studies show that this post-translational modification can be affected by the presence of a poor substrate such as moxalactam but also by the V117T substitution. Val117 is a strictly conserved hydrophobic residue located in the active site. In addition, inhibition of class D β-lactamases by chloride ions is due to a competition between the side chain carboxylate of the modified Lys70 and chloride ions. Determination of the individual kinetic constants shows that the deacylation of the acyl-enzyme is the rate-limiting step for the wild-type OXA-10 β-lactamase.     

Metagenomic era for biocatalyst identification

Microbial enzymes have many known applications as biocatalysts. However, only a few of them are currently employed for biocatalysis even though an annotated collection of more than 190 billion bases is available in metagenome sequence databases from uncultured and highly diverse microbial populations. This review aims at providing conceptual and technical bases for the translation of metagenome data into both experimental and computational frameworks that facilitates a comprehensive analysis of the biocatalysts diversity space. We will also briefly present the status of the current capabilities that assess and predict catalytic potential of environmental sites and track its diversity and evolution in large-scale biocatalysis process resulting from studies applying metagenomics in association with gene fingerprinting, catabolic arrays and complementary '-omics'.