The Microbial Rosetta Stone Database: A compilation of global and emerging infectious microorganisms and bioterrorist threat agents

BACKGROUND: Thousands of different microorganisms affect the health, safety, and economic stability of populations. Many different medical and governmental organizations have created lists of the pathogenic microorganisms relevant to their missions; however, the nomenclature for biological agents on these lists and pathogens described in the literature is inexact. This ambiguity can be a significant block to effective communication among the diverse communities that must deal with epidemics or bioterrorist attacks. RESULTS: We have developed a database known as the Microbial Rosetta Stone. The database relates microorganism names, taxonomic classifications, diseases, specific detection and treatment protocols, and relevant literature. The database structure facilitates linkage to public genomic databases. This paper focuses on the information in the database for pathogens that impact global public health, emerging infectious organisms, and bioterrorist threat agents. CONCLUSION: The Microbial Rosetta Stone is available at . The database provides public access to up-to-date taxonomic classifications of organisms that cause human diseases, improves the consistency of nomenclature in disease reporting, and provides useful links between different public genomic and public health databases

Comparative Analysis of Ribonuclease P RNA of the Planctomycetes

The planctomycetes, order Planctomycetales, are a distinct phylum of domain Bacteria. Genes encoding the RNA portion of ribonuclease P (RNase P) of some planctomycete members were sequenced and compared with existing database planctomycete sequences. rnpB gene sequences encoding RNase P RNA were generated by a conserved primer PCR strategy for Planctomyces brasiliensis, Planctomyces limnophilus, Pirellula marina, Pirellula staleyi strain ATCC 35122, Isosphaera pallida, one other Isosphaera strain, Gemmata obscuriglobus and three other strains of the Gemmata group. These sequences were aligned against reference bacterial sequences and secondary structures of corresponding RNase P RNAs deduced by a comparative approach. P12 helices were found to be highly variable in length, as were helices P16.1 and P19, when present. RNase P RNA secondary structures of Gemmata isolates were found to have unusual features relative to other planctomycetes, including a long P9 helix and an insert in the P13 helix not found in any other member of domain Bacteria. These unique features are consistent with other unusual properties of this genus, distinguishing it from other bacteria. Phylogenetic analyses indicate that relationships between planctomycetes derived from RNase P RNA are consistent with 16S rRNA-based analyses

Analysis of antibiotic resistance genes in multidrug-resistant acinetobacter sp. isolates from military and civilian patients treated at the Walter Reed Army Medical Center

Military medical facilities treating patients injured in Iraq and Afghanistan have identified a large number of multidrug-resistant (MDR) Acinetobacter baumannii isolates. In order to anticipate the impact of these pathogens on patient care, we analyzed the antibiotic resistance genes responsible for the MDR phenotype in Acinetobacter sp. isolates collected from patients at the Walter Reed Army Medical Center (WRAMC). Susceptibility testing, PCR amplification of the genetic determinants of resistance, and clonality were determined. Seventy-five unique patient isolates were included in this study: 53% were from bloodstream infections, 89% were resistant to at least three classes of antibiotics, and 15% were resistant to all nine antibiotics tested. Thirty-seven percent of the isolates were recovered from patients nosocomially infected or colonized at the WRAMC. Sixteen unique resistance genes or gene families and four mobile genetic elements were detected. In addition, this is the first report of blaOXA-58-like and blaPER-like genes in the U.S. MDR A. baumannii isolates with at least eight identified resistance determinants were recovered from 49 of the 75 patients. Molecular typing revealed multiple clones, with eight major clonal types being nosocomially acquired and with more than 60% of the isolates being related to three pan-European types. This report gives a “snapshot” of the complex genetic background responsible for antimicrobial resistance in Acinetobacter spp. from the WRAMC. Identifying genes associated with the MDR phenotype and defining patterns of transmission serve as a starting point for devising strategies to limit the clinical impact of these serious infections. © 2006, American Society for Microbiolog

Knowledge-based instantiation of full atomic detail into coarse-grain RNA 3D structural models

Motivation: The recent development of methods for modeling RNA 3D structures using coarse-grain approaches creates a need to bridge low- and high-resolution modeling methods. Although they contain topological information, coarse-grain models lack atomic detail, which limits their utility for some applications

Attenuation of loop-receptor interactions with pseudoknot formation

RNA tetraloops can recognize receptors to mediate long-range interactions in stable natural RNAs. In vitro selected GNRA tetraloop/receptor interactions are usually more ‘G/C-rich’ than their ‘A/U-rich’ natural counterparts. They are not as widespread in nature despite comparable biophysical and chemical properties. Moreover, while AA, AC and GU dinucleotide platforms occur in natural GAAA/11 nt receptors, the AA platform is somewhat preferred to the others. The apparent preference for ‘A/U-rich’ GNRA/receptor interactions in nature might stem from an evolutionary adaptation to avoid folding traps at the level of the larger molecular context. To provide evidences in favor of this hypothesis, several riboswitches based on natural and artificial GNRA receptors were investigated in vitro for their ability to prevent inter-molecular GNRA/receptor interactions by trapping the receptor sequence into an alternative intra-molecular pseudoknot. Extent of attenuation determined by native gel-shift assays and co-transcriptional assembly is correlated to the G/C content of the GNRA receptor. Our results shed light on the structural evolution of natural long-range interactions and provide design principles for RNA-based attenuator devices to be used in synthetic biology and RNA nanobiotechnology

Rapid Identification of Emerging Pathogens: Coronavirus

New surveillance approach can analyze >900 polymerase chain reactions per day

Global Surveillance of Emerging Influenza Virus Genotypes by Mass Spectrometry

Effective influenza surveillance requires new methods capable of rapid and inexpensive genomic analysis of evolving viral species for pandemic preparedness, to understand the evolution of circulating viral species, and for vaccine strain selection. We have developed one such approach based on previously described broad-range reverse transcription PCR/electrospray ionization mass spectrometry (RT-PCR/ESI-MS) technology.Analysis of base compositions of RT-PCR amplicons from influenza core gene segments (PB1, PB2, PA, M, NS, NP) are used to provide sub-species identification and infer influenza virus H and N subtypes. Using this approach, we detected and correctly identified 92 mammalian and avian influenza isolates, representing 30 different H and N types, including 29 avian H5N1 isolates. Further, direct analysis of 656 human clinical respiratory specimens collected over a seven-year period (1999-2006) showed correct identification of the viral species and subtypes with >97% sensitivity and specificity. Base composition derived clusters inferred from this analysis showed 100% concordance to previously established clades. Ongoing surveillance of samples from the recent influenza virus seasons (2005-2006) showed evidence for emergence and establishment of new genotypes of circulating H3N2 strains worldwide. Mixed viral quasispecies were found in approximately 1% of these recent samples providing a view into viral evolution.Thus, rapid RT-PCR/ESI-MS analysis can be used to simultaneously identify all species of influenza viruses with clade-level resolution, identify mixed viral populations and monitor global spread and emergence of novel viral genotypes. This high-throughput method promises to become an integral component of influenza surveillance

Molecular modelling of the GIR1 branching ribozyme gives new insight into evolution of structurally related ribozymes

Twin-ribozyme introns contain a branching ribozyme (GIR1) followed by a homing endonuclease (HE) encoding sequence embedded in a peripheral domain of a group I splicing ribozyme (GIR2). GIR1 catalyses the formation of a lariat with 3 nt in the loop, which caps the HE mRNA. GIR1 is structurally related to group I ribozymes raising the question about how two closely related ribozymes can carry out very different reactions. Modelling of GIR1 based on new biochemical and mutational data shows an extended substrate domain containing a GoU pair distinct from the nucleophilic residue that dock onto a catalytic core showing a different topology from that of group I ribozymes. The differences include a core J8/7 region that has been reduced and is complemented by residues from the pre-lariat fold. These findings provide the basis for an evolutionary mechanism that accounts for the change from group I splicing ribozyme to the branching GIR1 architecture. Such an evolutionary mechanism can be applied to other large RNAs such as the ribonuclease P

Cleavage mediated by the P15 domain of bacterial RNase P RNA

Independently folded domains in RNAs frequently adopt identical tertiary structures regardless of whether they are in isolation or are part of larger RNA molecules. This is exemplified by the P15 domain in the RNA subunit (RPR) of the universally conserved endoribonuclease P, which is involved in the processing of tRNA precursors. One of its domains, encompassing the P15 loop, binds to the 3′-end of tRNA precursors resulting in the formation of the RCCA–RNase P RNA interaction (interacting residues underlined) in the bacterial RPR–substrate complex. The function of this interaction was hypothesized to anchor the substrate, expose the cleavage site and result in re-coordination of Mg2+ at the cleavage site. Here we show that small model-RNA molecules (~30 nt) carrying the P15-loop mediated cleavage at the canonical RNase P cleavage site with significantly reduced rates compared to cleavage with full-size RPR. These data provide further experimental evidence for our model that the P15 domain contributes to both substrate binding and catalysis. Our data raises intriguing evolutionary possibilities for ‘RNA-mediated’ cleavage of RNA