VDNA: The virtual DNA plug-in for VMD

Summary: The DNA inter base pair step parameters (Tilt, Roll, Twist, Shift, Slide, Rise) are a standard internal coordinate representation of DNA. In the absence of bend and shear, it is relatively easy to mentally visualize how Twist and Rise generate the familiar double helix. More complex structures do not readily yield to such intuition. For this reason, we developed a plug-in for VMD that accepts a set of mathematical expressions as input and generates a coarse-grained model of DNA as output. This feature of VDNA appears to provide a unique approach to DNA modeling. Predefined expressions include: linear, sheared, bent and circular DNA, and models of the nucleosome superhelix, chromatin, thermal motion and nucleosome unwrapping

MADNet: microarray database network web server

MADNet is a user-friendly data mining and visualization tool for rapid analysis of diverse high-throughput biological data such as microarray, phage display or even metagenome experiments. It presents biological information in the context of metabolic and signalling pathways, transcription factors and drug targets through minimal user input, consisting only of the file with the experimental data. These data are integrated with information stored in various biological databases such as NCBI nucleotide and protein databases, metabolic and signalling pathway databases (KEGG), transcription regulation (TRANSFAC©) and drug target database (DrugBank). MADNet is freely available for academic use at http://www.bioinfo.hr/madnet

Transcriptional Regulation of the Capsular Polysaccharide Biosynthesis Locus of Streptococcus Pneumoniae: a Bioinformatic Analysis

The polysaccharide capsule of Streptococcus pneumoniae is the main virulence factor, which makes the bacterium resistant to phagocytosis. Expression of capsular polysaccharide must be adjusted at different stages of pneumococcal infection, thus, their transcriptional regulation appears to be crucial. To get insight into the existence of regulatory mechanisms common to most serotypes, a bioinformatic analysis of the DNA region located upstream of the capsular locus was performed. With the exception of serotype 37, the capsular locus is located between dexB and aliA on the pneumococcal chromosome. Up to 26 different sequence organizations were found among pneumococci synthesizing their capsule through a Wzy-polymerase-dependent mechanism, mostly varying according to the presence/absence of distinct insertion elements. As a consequence, only ∼250 bp (including a 107 bp RUP_A element) was conserved in 86 sequences, although only a short (ca. 87 bp) region located immediately upstream of cpsA was strictly conserved in all the sequences analyzed. An exhaustive search for possible operator sequences was done. Interestingly, although the promoter region of serotype 3 isolates completely differs from that of other serotypes, most of the proteins proposed to regulate transcription in serotype 3 pneumococci were also predicted to function as possible regulators in non-serotype 3 S. pneumoniae isolates

Determination of the target nucleosides for members of two families of 16S rRNA methyltransferases that confer resistance to partially overlapping groups of aminoglycoside antibiotics

The 16S ribosomal RNA methyltransferase enzymes that modify nucleosides in the drug binding site to provide self-resistance in aminoglycoside-producing micro-organisms have been proposed to comprise two distinct groups of S-adenosyl-l-methionine (SAM)-dependent RNA enzymes, namely the Kgm and Kam families. Here, the nucleoside methylation sites for three Kgm family methyltransferases, Sgm from Micromonospora zionensis, GrmA from Micromonospora echinospora and Krm from Frankia sp. Ccl3, were experimentally determined as G1405 by MALDI-ToF mass spectrometry. These results significantly extend the list of securely characterized G1405 modifying enzymes and experimentally validate their grouping into a single enzyme family. Heterologous expression of the KamB methyltransferase from Streptoalloteichus tenebrarius experimentally confirmed the requirement for an additional 60 amino acids on the deduced KamB N-terminus to produce an active methyltransferase acting at A1408, as previously suggested by an in silico analysis. Finally, the modifications at G1405 and A1408, were shown to confer partially overlapping but distinct resistance profiles in Escherichia coli. Collectively, these data provide a more secure and systematic basis for classification of new aminoglycoside resistance methyltransferases from producers and pathogenic bacteria on the basis of their sequences and resistance profiles

Structural insights into the function of aminoglycoside-resistance A1408 16S rRNA methyltransferases from antibiotic-producing and human pathogenic bacteria

X-ray crystal structures were determined of the broad-spectrum aminoglycoside-resistance A1408 16S rRNA methyltransferases KamB and NpmA, from the aminoglycoside-producer Streptoalloteichus tenebrarius and human pathogenic Escherichia coli, respectively. Consistent with their common function, both are Class I methyltransferases with additional highly conserved structural motifs that embellish the core SAM-binding fold. In overall structure, the A1408 rRNA methyltransferase were found to be most similar to a second family of Class I methyltransferases of distinct substrate specificity (m7G46 tRNA). Critical residues for A1408 rRNA methyltransferase activity were experimentally defined using protein mutagenesis and bacterial growth assays with kanamycin. Essential residues for SAM coenzyme binding and an extended protein surface that likely interacts with the 30S ribosomal subunit were thus revealed. The structures also suggest potential mechanisms of A1408 target nucleotide selection and positioning. We propose that a dynamic extended loop structure that is positioned adjacent to both the bound SAM and a functionally critical structural motif may mediate concerted conformational changes in rRNA and protein that underpin the specificity of target selection and activation of methyltransferase activity. These new structures provide important new insights that may provide a starting point for strategies to inhibit these emerging causes of pathogenic bacterial resistance to aminoglycosides

Insights into the structure, function and evolution of the radical-SAM 23S rRNA methyltransferase Cfr that confers antibiotic resistance in bacteria

The Cfr methyltransferase confers combined resistance to five classes of antibiotics that bind to the peptidyl tranferase center of bacterial ribosomes by catalyzing methylation of the C-8 position of 23S rRNA nucleotide A2503. The same nucleotide is targeted by the housekeeping methyltransferase RlmN that methylates the C-2 position. Database searches with the Cfr sequence have revealed a large group of closely related sequences from all domains of life that contain the conserved CX3CX2C motif characteristic of radical S-adenosyl-l-methionine (SAM) enzymes. Phylogenetic analysis of the Cfr/RlmN family suggests that the RlmN subfamily is likely the ancestral form, whereas the Cfr subfamily arose via duplication and horizontal gene transfer. A structural model of Cfr has been calculated and used as a guide for alanine mutagenesis studies that corroborate the model-based predictions of a 4Fe–4S cluster, a SAM molecule coordinated to the iron–sulfur cluster (SAM1) and a SAM molecule that is the putative methyl group donor (SAM2). All mutations at predicted functional sites affect Cfr activity significantly as assayed by antibiotic susceptibility testing and primer extension analysis. The investigation has identified essential amino acids and Cfr variants with altered reaction mechanisms and represents a first step towards understanding the structural basis of Cfr activity

Demosponge EST Sequencing Reveals a Complex Genetic Toolkit of the Simplest Metazoans

Sponges (Porifera) are among the simplest living and the earliest branching metazoans. They hold a pivotal role for studying genome evolution of the entire metazoan branch, both as an outgroup to Eumetazoa and as the closest branching phylum to the common ancestor of all multicellular animals (Urmetazoa). In order to assess the transcription inventory of sponges, we sequenced expressed sequence tag libraries of two demosponge species, Suberites domuncula and Lubomirskia baicalensis, and systematically analyzed the assembled sponge transcripts against their homologs from complete proteomes of six well-characterized metazoans—Nematostella vectensis, Caenorhabditis elegans, Drosophila melanogaster, Strongylocentrotus purpuratus, Ciona intestinalis, and Homo sapiens. We show that even the earliest metazoan species already have strikingly complex genomes in terms of gene content and functional repertoire and that the rich gene repertoire existed even before the emergence of true tissues, therefore further emphasizing the importance of gene loss and spatio-temporal changes in regulation of gene expression in shaping the metazoan genomes. Our findings further indicate that sponge and human genes generally show similarity levels higher than expected from their respective positions in metazoan phylogeny, providing direct evidence for slow rate of evolution in both “basal” and “apical” metazoan genome lineages. We propose that the ancestor of all metazoans had already had an unusually complex genome, thereby shifting the origins of genome complexity from Urbilateria to Urmetazoa

Structural basis for the methylation of A1408 in 16S rRNA by a panaminoglycoside resistance methyltransferase NpmA from a clinical isolate and analysis of the NpmA interactions with the 30S ribosomal subunit

NpmA, a methyltransferase that confers resistance to aminoglycosides was identified in an Escherichia coli clinical isolate. It belongs to the kanamycin–apramycin methyltransferase (Kam) family and specifically methylates the 16S rRNA at the N1 position of A1408. We determined the structures of apo-NpmA and its complexes with S-adenosylmethionine (AdoMet) and S-adenosylhomocysteine (AdoHcy) at 2.4, 2.7 and 1.68 Å, respectively. We generated a number of NpmA variants with alanine substitutions and studied their ability to bind the cofactor, to methylate A1408 in the 30S subunit, and to confer resistance to kanamycin in vivo. Residues D30, W107 and W197 were found to be essential. We have also analyzed the interactions between NpmA and the 30S subunit by footprinting experiments and computational docking. Helices 24, 42 and 44 were found to be the main NpmA-binding site. Both experimental and theoretical analyses suggest that NpmA flips out the target nucleotide A1408 to carry out the methylation. NpmA is plasmid-encoded and can be transferred between pathogenic bacteria; therefore it poses a threat to the successful use of aminoglycosides in clinical practice. The results presented here will assist in the development of specific NpmA inhibitors that could restore the potential of aminoglycoside antibiotics