Phenotypic and genotypic characterization of Klebsiella pneumoniae isolates recovered from nonhuman primates

Klebsiella pneumoniae is a zoonotic, Gram-negative member of the family Enterobacteriaceae and is the causative agent of nosocomial septicemic, pneumonic, and urinary tract infections. Recently, pathogenic strains of K. pneumoniae sharing a hypermucoviscosity (HMV) phenotype have been attributed to multisystemic abscessation in both human and nonhuman primates. Although K. pneumoniae is a well-recognized zoonotic agent, there is a lack of general information including adequate diagnostic methods or treatments for nonhuman primates. In an effort to increase the body of knowledge of this enigmatic pathogen, K. pneumoniae isolates from African green monkeys (Chlorocebus aethiops sabaeus) on the island of St. Kitts, West Indies were genotypically and phenotypically characterized. Genetic fingerprints generated by PCR-mediated genomic fingerprinting, phenotypic characterization, and antimicrobial susceptibility all identified a high degree of similarity between the HMV and non-HMV K. pneumoniae isolates. The results obtained from this work will help establish a baseline for the development of efficacious diagnostic methods and treatment strategies for both human and nonhuman primates.     

The dimanganese(II) site of Bacillus subtilis class Ib ribonucleotide reductase

Class Ib ribonucleotide reductases (RNRs) use a dimanganese-tyrosyl radical cofactor, Mn(III)(2)-Y(?), in their homodimeric NrdF (β2) subunit to initiate reduction of ribonucleotides to deoxyribonucleotides. The structure of the Mn(II)(2) form of NrdF is an important component in understanding O(2)-mediated formation of the active metallocofactor, a subject of much interest because a unique flavodoxin, NrdI, is required for cofactor assembly. Biochemical studies and sequence alignments suggest that NrdF and NrdI proteins diverge into three phylogenetically distinct groups. The only crystal structure to date of a NrdF with a fully ordered and occupied dimanganese site is that of Escherichia coli Mn(II)(2)-NrdF, prototypical of the enzymes from actinobacteria and proteobacteria. Here we report the 1.9 ? resolution crystal structure of Bacillus subtilis Mn(II)(2)-NrdF, representative of the enzymes from a second group, from Bacillus and Staphylococcus. The structures of the metal clusters in the β2 dimer are distinct from those observed in E. coli Mn(II)(2)-NrdF. These differences illustrate the key role that solvent molecules and protein residues in the second coordination sphere of the Mn(II)(2) cluster play in determining conformations of carboxylate residues at the metal sites and demonstrate that diverse coordination geometries are capable of serving as starting points for Mn(III)(2)-Y(?) cofactor assembly in class Ib RNRs.     

Phasing of many thousands of genotyped samples

Haplotypes are an important resource for a large number of applications in human genetics, but computationally inferred haplotypes are subject to switch errors that decrease their utility. The accuracy of computationally inferred haplotypes increases with sample size, and although ever larger genotypic data sets are being generated, the fact that existing methods require substantial computational resources limits their applicability to data sets containing tens or hundreds of thousands of samples. Here, we present HAPI-UR (haplotype inference for unrelated samples), an algorithm that is designed to handle unrelated and/or trio and duo family data, that has accuracy comparable to or greater than existing methods, and that is computationally efficient and can be applied to 100,000 samples or more. We use HAPI-UR to phase a data set with 58,207 samples and show that it achieves practical runtime and that switch errors decrease with sample size even with the use of samples from multiple ethnicities. Using a data set with 16,353 samples, we compare HAPI-UR to Beagle, MaCH, IMPUTE2, and SHAPEIT and show that HAPI-UR runs 18× faster than all methods and has a lower switch-error rate than do other methods except for Beagle; with the use of consensus phasing, running HAPI-UR three times gives a slightly lower switch-error rate than Beagle does and is more than six times faster. We demonstrate results similar to those from Beagle on another data set with a higher marker density. Lastly, we show that HAPI-UR has better runtime scaling properties than does Beagle so that for larger data sets, HAPI-UR will be practical and will have an even larger runtime advantage. HAPI-UR is available online (see Web Resources).     

Novel High-Molecular-Weight,R-Type Bacteriocins of Clostridium difficile

Clostridium difficile causes one of the leading nosocomial infections in developed countries, and therapeutic choices are limited. Some strains of C. difficile produce phage tail-like particles upon induction of the SOS response. These particles have bactericidal activity against other C. difficile strains and can therefore be classified as bacteriocins, similar to the R-type pyocins of Pseudomonas aeruginosa. These R-type bacteriocin particles, which have been purified from different strains, each have a different C. difficile-killing spectrum, with no one bacteriocin killing all C. difficile isolates tested. We have identified the genetic locus of these “diffocins” (open reading frames 1359 to 1376) and have found them to be common among the species. The entire diffocin genetic locus of more than 20 kb was cloned and expressed in Bacillus subtilis, and this resulted in production of bactericidal particles. One of the interesting features of these particles is a very large structural protein of ∼200 kDa, the product of gene 1374. This large protein determines the killing spectrum of the particles and is likely the receptor-binding protein. Diffocins may provide an alternate bactericidal agent to prevent or treat infections and to decolonize individuals who are asymptomatic carriers.     

Assembly and dynamics of Gp59-Gp32-single-stranded DNA (ssDNA), a DNA helicase loading complex required for recombination-dependent replication in bacteriophage T4

The Gp59 protein of bacteriophage T4 plays critical roles in recombination-dependent DNA replication and repair by correctly loading the replicative helicase, Gp41, onto recombination intermediates. Previous work demonstrated that Gp59 is required to load helicase onto single-stranded DNA that is saturated with Gp32, the T4 single-stranded DNA (ssDNA)-binding protein. Gp59 and Gp32 bind simultaneously to ssDNA, forming a Gp59-Gp32-ssDNA complex that is a key intermediate in helicase loading. Here we characterize the assembly and dynamics of this helicase loading complex (HLC) through changes in the fluorescent states of Gp32F, a fluorescein-Gp32 conjugate. Results show that HLC formation requires a minimum Gp32-ssDNA cluster size and that Gp59 co-localizes with Gp32-ssDNA clusters in the presence of excess free ssDNA. These and other results indicate that Gp59 targets helicase assembly onto Gp32-ssDNA clusters that form on the displaced strand of D-loops, which suggests a mechanism for the rapid initiation of recombination-dependent DNA replication. Helicase loading at the HLC requires ATP binding (not hydrolysis) by Gp41 and results in local remodeling of Gp32 within the HLC. Subsequent ATPase-driven translocation of Gp41 progressively disrupts Gp32-ssDNA interactions. Evidence suggests that Gp59 from the HLC is recycled to promote multiple rounds of helicase assembly on Gp32-ssDNA, a capability that could be important for the restart of stalled replication forks.     

Probing binding pocket of serotonin transporter by single molecular force spectroscopy on living cells

The serotonin transporter (SERT) terminates neurotransmission by removing serotonin from the synaptic cleft. In addition, it is the site of action of antidepressants (which block the transporter) and of amphetamines (which induce substrate efflux). The interaction energies involved in binding of such compounds to the transporter are unknown. Here, we used atomic force microscopy (AFM) to probe single molecular interactions between the serotonin transporter and MFZ2-12 (a potent cocaine analog) in living CHOK1 cells. For the AFM measurements, MFZ2-12 was immobilized on AFM tips by using a heterobifunctional cross-linker. By varying the pulling velocity in force distance cycles drug-transporter complexes were ruptured at different force loadings allowing for mapping of the interaction energy landscape. We derived chemical rate constants from these recordings and compared them with those inferred from inhibition of transport and ligand binding: koff values were in good agreement with those derived from uptake experiments; in contrast, the kon values were scaled down when determined by AFM. Our observations generated new insights into the energy landscape of the interaction between SERT and inhibitors. They thus provide a useful framework for molecular dynamics simulations by exploring the range of forces and energies that operate during the binding reaction.     

Crystal structure and RNA binding properties of the RNA recognition motif (RRM) and AlkB domains in human AlkB homolog 8 (ABH8), an enzyme catalyzing tRNA hypermodification

Humans express nine paralogs of the bacterial DNA repair enzyme AlkB, an iron/2-oxoglutarate-dependent dioxygenase that reverses alkylation damage to nucleobases. The biochemical and physiological roles of these paralogs remain largely uncharacterized, hampering insight into the evolutionary expansion of the AlkB family. However, AlkB homolog 8 (ABH8), which contains RNA recognition motif (RRM) and methyltransferase domains flanking its AlkB domain, recently was demonstrated to hypermodify the anticodon loops in some tRNAs. To deepen understanding of this activity, we performed physiological and biophysical studies of ABH8. Using GFP fusions, we demonstrate that expression of the Caenorhabditis elegans ABH8 ortholog is widespread in larvae but restricted to a small number of neurons in adults, suggesting that its function becomes more specialized during development. In vitro RNA binding studies on several human ABH8 constructs indicate that binding affinity is enhanced by a basic α-helix at the N terminus of the RRM domain. The 3.0-Å-resolution crystal structure of a construct comprising the RRM and AlkB domains shows disordered loops flanking the active site in the AlkB domain and a unique structural Zn(II)-binding site at its C terminus. Although the catalytic iron center is exposed to solvent, the 2-oxoglutarate co-substrate likely adopts an inactive conformation in the absence of tRNA substrate, which probably inhibits uncoupled free radical generation. A conformational change in the active site coupled to a disorder-to-order transition in the flanking protein segments likely controls ABH8 catalytic activity and tRNA binding specificity. These results provide insight into the functional and structural adaptations underlying evolutionary diversification of AlkB domains.