Functional assembly of nitrous oxide reductase provides insights into copper site maturation

The multicopper enzyme nitrous oxide reductase reduces the greenhouse gas N2O to uncritical N2as the final step of bacterial denitrification. Its two metal centers require an elaborate assembly machinery that so far has precluded heterologous production as a prerequisite for bioremediatory applications in agriculture and wastewater treatment. Here, we report on the production of active holoenzyme inEscherichia coliusing a two-plasmid system to produce the entire biosynthetic machinery as well as the structural gene for the enzyme. Using this recombinant system to probe the role of individual maturation factors, we find that the ABC transporter NosFY and the accessory NosD protein are essential for the formation of the [4Cu:2S] site CuZ, but not the electron transfer site CuA. Depending on source organism, the heterologous hostE. colican, in some cases, compensate for the lack of the Cu chaperone NosL, while in others this protein is strictly required, underlining the case for designing a recombinant system to be entirely self-contained.

The importance of Chargaff’s second parity rule for genomic signatures in metagenomics

An important problem in metagenomic data analysis is to identify the source organism, or at least taxon, of each sequence. Most methods tackle this problem in two steps by using an alignment-free approach: first the DNA sequences are represented as points of a real n-dimensional space via a mapping function then either clustering or classification algorithms are applied. Those mapping functions require to be genomic signatures: the dissimilarity between the mapped points must reflect the degree of phylogenetic similarity of the source species. Designing good signatures for metagenomics can be challenging due to the special characteristics of metagenomic sequences; most of the existing signatures were not designed accordingly and they were tested only on error-free sequences sampled from a few dozens of species.In this work we analyze comparatively the goodness of existing and novel signatures based on tetranu-cleotide frequencies via statistical models and computational experiments; we also study how they are affected by the generalized Chargaff’s second parity rule (GCSPR), which states that in a given sequence longer than 50kbp, inverse oligonucleotides are approximately equally frequent. We analyze 38 million sequences of 150 bp-1,000 bp with 1% base-calling error, sampled from 1,284 microbes. Our models indicate that GCSPR reduces strand-dependence of signatures, that is, their values are less affected by the source strand; GCSPR is further exploited by some signatures to reduce the intra-species dispersion. Two novel signatures stand out both in the models and in the experiments: the combination signature and the operation signature. The former achieves strand-independence without grouping oligonucleotides; this could be valuable for alignment-free sequence comparison methods when distinguishing inverse oligonucleotides matters. Operation signature sums the frequencies of reverse, complement, and inverse tetranucleotides; having 72 features it reduces the computational intensity of the analysis.

Pengembangan Database Genbank UAI-Bioinformatics Menggunakan Sistem Terdistribusi

<p class="Default"><em>Abstrak</em> – <strong>T</strong><strong>elah dilakukan penelitian tentang </strong><strong>pengolahan terdistribusi data genbank menggunakan <em>Hadoop Distributed Filesystem </em>(HDFS) dengan tujuan mengetahui efektifitas pengolahan data genbank khususnya pada pencarian sequens dengan data masukan yang berukuran besar.</strong><strong> Penelitian dilakukan di </strong><strong>L</strong><strong>aboratorium </strong><strong>Jaringan Universitas Al Azhar Indonesia dengan menggunakan 6 komputer dan satu <em>server</em> dimana dalam <em>Hadoop</em> menjadi 7 <em>node</em> dengan rincian 1 <em>namenode</em>, 7 <em>datanode</em>, 1 secondary <em>namenode</em>. Dengan eksperimen HDFS menggunakan 1 <em>node</em>, 2 <em>node</em>, 4 <em>node</em>, 6 <em>node</em>, dan 7 <em>node</em> dibandingkan dengan <em>Local Filesystem</em>. Hasil menunjukan proses pencarian sequens data genbank menggunakan 1 – 7 <em>node</em> pada skenario eksperimen pertama dengan <em>output</em> yang menampilkan hasil 3 <em>field</em> <em>(Locus, Definition, </em>dan<em> Authors</em>), skenario eksperimen kedua dengan <em>output</em> yang menampilkan hasil 3 <em>field</em> <em>(Locus, Authors, </em>dan<em> Origin)</em>, dan skenario eksperimen ketiga menggunakan HDFS dan LFS dengan <em>output</em> yang menampilkan seluruh <em>field</em> yang terdapat dalam data genbank (</strong><strong><em>Locus, Definition, Accesion, Version, Keywords, Source, Organism, Reference, Authors, Title, Journal, Pubmed, Comment, Features, </em></strong><strong>dan<em> Origin</em></strong><strong>). Evaluasi menunjukan bahwa proses pencarian sequens data genbank menggunakan HDFS dengan 7 <em>node</em> adalah 4 kali lebih cepat dibandingkan dengan menggunakan 1 <em>node</em>. Sedangkan perbedaan waktu pada penggunaan HDFS dengan 1 <em>node</em> adalah 1.02 kali lebih cepat dibandingkan dengan <em>Local Filesystem</em> dengan 4 <em>core</em> <em>processor</em>.</strong></p><p class="Default"><strong> </strong></p><p><em>Abstract </em><strong>- A research on distributed processing of GenBank data using Hadoop Distributed File System GenBank (HDFS) in order to know the effectiveness of data processing, especially in the search sequences with large input data. Research conducted at the Network Laboratory of the University of Al Azhar Indonesia using 6 computers and a server where the Hadoop to 7 nodes with details 1 namenode, 7 datanode, 1 secondary namenode. With HDFS experiments using 1 node, node 2, node 4, node 6, and 7 nodes compared with the Local Filesystem. The results show the search process of data GenBank sequences using 1-7 nodes in the first experiment scenario with an output that displays the results of 3 fields (Locus, Definition, and Authors), a second experiment scenario with an output that displays the results of 3 fields (Locus, Authors, and Origin) , and the third experiment scenarios using HDFS and LFS with output that displays all the data fields contained in GenBank (Locus, Definition, Accesion, Version, Keywords, Source, Organism, Reference, Authors, Title, Journal, Pubmed, Comment, Features, and Origin). Evaluation shows that the search process of data GenBank sequences using HDFS with 7 nodes is 4 times faster than using one node. While the time difference in the use of HDFS with one node is 1:02 times faster than the Local File System with 4 core processor.</strong></p><p><strong><em> </em></strong></p><p><strong><em></em></strong><strong><em>Keywords </em></strong><em>– genbank, sequens, distributed computing, Hadoop, HDFS</em></p>

Network Analysis of Plasmidomes: The Azospirillum brasilense Sp245 Case

Azospirillum brasilense is a nitrogen-fixing bacterium living in association with plant roots. The genome of the strain Sp245, isolated in Brazil from wheat roots, consists of one chromosome and six plasmids. In this work, the A. brasilense Sp245 plasmids were analyzed in order to shed some light on the evolutionary pathways they followed over time. To this purpose, a similarity network approach was applied in order to identify the evolutionary relationships among all the A. brasilense plasmids encoded proteins; in this context a computational pipeline specifically devoted to the analysis and the visualization of the network-like evolutionary relationships among different plasmids molecules was developed. This information was supplemented with a detailed (in silico) functional characterization of both the connected (i.e., sharing homology with other sequences in the dataset) and the unconnected (i.e., not sharing homology) components of the network. Furthermore, the most likely source organism for each of the genes encoded by A. brasilense plasmids was checked, allowing the identification of possible trends of gene loss/gain in this microorganism. Data obtained provided a detailed description of the evolutionary landscape of the plasmids of A. brasilense Sp245, suggesting some of the molecular mechanisms responsible for the present-day structure of these molecules.

Monitoring protein precipitates by in-house X-ray powder diffraction

Powder diffraction from protein powders using in-house diffractometers is an effective tool for identification and monitoring of protein crystal forms and artifacts. As an alternative to conventional powder diffractometers a single crystal diffractometer equipped with an X-ray micro-source can be used to collect powder patterns from 1 µl samples. Using a small-angle X-ray scattering (SAXS) camera it is possible to collect data within minutes. A streamlined program has been developed for the calculation of powder patterns from pdb-coordinates, and includes correction for bulk-solvent. A number of such calculated powder patterns from insulin and lysozyme have been included in the powder diffraction database and successfully used for search-match identification. However, the fit could be much improved if peak asymmetry and multiple bulk-solvent corrections were included. When including a large number of protein data sets in the database some problems can be foreseen due to the large number of overlapping peaks in the low-angle region, and small differences in unit cell parameters between pdb-data and powder data. It is suggested that protein entries are supplied with more searchable keywords as protein name, protein type, molecular weight, source organism etc. in order to limit possible hits.

Detection of Ehrlichia risticii, the Agent of Potomac Horse Fever, in Freshwater Stream Snails (Pleuroceridae:Juga spp.) from Northern California

ABSTRACT Ehrlichia DNA was identified by nested PCR in operculate snails (Pleuroceridae: Juga spp.) collected from stream water in a northern California pasture in which Potomac horse fever (PHF) is enzootic. Sequencing of PCR-amplified DNA from a suite of genes (the 16S rRNA, groESL heat shock operon, 51-kDa major antigen genes) indicated that the source organism closely resembledEhrlichia risticii, the causative agent of PHF. The minimum percentage of Juga spp. harboring the organism in the population studied was 3.5% (2 of 57 snails). No ehrlichia DNA was found in tissues of 123 lymnaeid, physid, and planorbid snails collected at the same site. These data suggest that pleurocerid stream snails may play a role in the life cycle of E. risticii in northern California.

Production and Characterization of Ehrlichia risticii, the Agent of Potomac Horse Fever, from Snails (Pleuroceridae: Juga spp.) in Aquarium Culture and Genetic Comparison to Equine Strains

We report on the production and characterization of Ehrlichia risticii, the agent of Potomac horse fever (PHF), from snails (Pleuroceridae: Juga spp.) maintained in aquarium culture and compare it genetically to equine strains. Snails were collected from stream waters on a pasture in Siskiyou County, Calif., where PHF is enzootic and were maintained for several weeks in freshwater aquaria in the laboratory. Upon exposure to temperatures above 22°C the snails released trematode cercariae tentatively identified as virgulate cercariae. Fragments of three different genes (genes for 16S rRNA, thegroESL heat shock operon, and the 51-kDa major antigen) were amplified from cercaria lysates by PCR and sequenced. Genetic information was also obtained from E. risticii strains from horses with PHF. The PCR positivity of snail secretions was associated with the presence of trematode cercariae. Sequence analysis of the three genes indicated that the source organism closely resembledE. risticii, and the sequences of all three genes were virtually identical to those of the genes of an equine E. risticii strain from a property near the snail collection site. Phylogenetic analyses of the three genes indicated the presence of geographical E. risticii strain clusters.