A phylogeny of bacterial RNA nucleotidyltransferases: Bacillus halodurans contains two tRNA nucleotidyltransferases

We have analyzed the distribution of RNA nucleotidyltransferases from the family that includes poly(A) polymerases (PAP) and tRNA nucleotidyltransferases (TNT) in 43 bacterial species. Genes of several bacterial species encode only one member of the nucleotidyltransferase superfamily (NTSF), and if that protein functions as a TNT, those organisms may not contain a poly(A) polymerase I like that of Escherichia coli. The genomes of several of the species examined encode more than one member of the nucleotidyltransferase superfamily. The function of some of those proteins is known, but in most cases no biochemical activity has been assigned to the NTSF. The NTSF protein sequences were used to construct an unrooted phylogenetic tree. To learn more about the function of the NTSFs in species whose genomes encode more than one, we have examined Bacillus halodurans. We have demonstrated that B. halodurans adds poly(A) tails to the 3' ends of RNAs in vivo. We have shown that the genes for both of the NTSFs encoded by the B. halodurans genome are transcribed in vivo. We have cloned, overexpressed, and purified the two NTSFs and have shown that neither functions as poly(A) polymerase in vitro. Rather, the two proteins function as tRNA nucleotidyltransferases, and our data suggest that, like some of the deep branching bacterial species previously studied by others, B. halodurans possesses separate CC- and A-adding tRNA nucleotidyltransferases. These observations raise the interesting question of the identity of the enzyme responsible for RNA polyadenylation in Bacillus.     

Biogeography and divergence times in the mulberry family (Moraceae)

The biogeographical history of the mulberry family (Moraceae) was investigated using phylogenetic inferences from nuclear and chloroplast DNA, molecular dating with multiple fossil calibrations, and independent geological evidence. The Moraceae are centered in the tropics which has invited the hypothesis that the family has Gondwanan origins and extant distribution is the result of vicariance due to the break-up of Gondwana. However, the cosmopolitan distribution of Moraceae suggests a more complicated biogeographical history. The timing and location of Moraceae diversification also bears on the origin of the fig pollination mutualism, a model for the study of coevolution and specialization. Recent molecular dating of pollinating fig wasps suggested that an ancient Gondwanan origin coupled with vicariance and dispersal could account for the present day distribution of the mutualism. Here, we provide the first assessment of this hypothesis based on dating of figs and their relatives. Minimum age estimates suggest that the Moraceae had diversified by at least the mid-Cretaceous and major clades including the figs may have radiated during the Tertiary after the break-up of Gondwanaland. Molecular evidence together with Eurasian fossils suggest that the early diversification of Moraceae in Eurasia and subsequent migration into the southern hemisphere is at least as plausible as the Gondwanan hypothesis. These findings invite a reevaluation of the biogeography of fig pollination and highlight the need for incorporating multiple sources of evidence in biogeographical reconstructions.     

Loop movement and catalysis in creatine kinase

Recently the crystal structure of creatine kinase from Torpedocalifornica was determined to 2.1 A. The dimeric structure revealed two different forms in the unit cell: one monomer was bound to a substrate, MgADP, and the other monomer was bound to a transition-state analogue complex composed of MgADP, nitrate and creatine. The most striking difference between the structures is the movement of two loops (comprising residues 60-70 and residues 323-333) into the active site in the transition state structure. This loop movement effectively occludes the active site from solvent, and the loops appear to be locked into place by a salt bridge formed between His66 and Asp326. His66 is of particular interest as it is located within a PGHP motif conserved in all creatine kinases but not found in other guanidino kinases. We have carried out alanine-scanning mutagenesis of each of the residues in the PGHP motif and determined that only the His66 plays a significant role in the creatine kinase reaction. Although neither residue interacts directly with the substrate, the interaction His66 and Asp326 appears to be important in providing the precise alignment of substrates necessary for phosphoryl group transfer. Finally, it is clear that neither His66 nor Asp326 are responsible for the pKs observed in the pH-rate profile for HMCK.     

Requirements for chemotaxis protein localization in Rhodobacter sphaeroides

Many proteins have recently been shown to localize to different regions of the bacterial cell. This is most striking in the case of the Escherichia coli chemotaxis pathway in which the components localize at the cell poles. Rhodobacter sphaeroides has a more complex chemotaxis system with two complete pathways, each localizing to different positions, one pathway at the pole and one at a discrete cluster within the cytoplasm of the bacterium. Using genomic replacement of the wild-type chemotaxis genes in R. sphaeroides with their corresponding fluorescent protein fusions in conjunction with in frame deletions of other chemotaxis genes, we have investigated which proteins are required for the formation of the polar and cytoplasmic chemotaxis protein clusters. As in E. coli, the polarly targeted CheA and CheW homologues are required for the formation of the polar cluster. However, the formation of the cytoplasmic cluster requires the cytoplasmic chemoreceptors and CheW but not the CheAs. Interestingly, even when deletion of a component resulted in the chemotaxis proteins of one pathway becoming delocalized and diffuse in the cytoplasm, in no case were any chemotaxis proteins seen to localize to the other signalling cluster.     

Hypoxia Moderates γ(1)34.5-Deleted Herpes Simplex Virus Oncolytic Activity in Human Glioma Xenoline Primary Cultures

Hypoxia plays a critical role in the tumor microenvironment of high-grade gliomas by promoting the glioma stem cell (GSC)-like phenotype, which displays resistance to standard therapies. We tested three glioblastoma multiforme xenograft lines (xenolines) against γ(1)34.5-deleted recombinant oncolytic herpes simplex virus (oHSV) C101 under 1% (hypoxia) and 20.8% (normoxia) oxygen tension for effects on oHSV infectivity, replication, and cytotoxicity in all tumor cells and CD133(+) GSCs. Expression levels of CD133, a putative GSC marker, and CD111 (nectin-1), an adhesion molecule that is the most efficient method for HSV entry, increased significantly under hypoxia in all three xenolines. Despite increased CD111 expression under hypoxic conditions, oHSV infectivity, cytotoxicity and viral recovery were not improved or were diminished in all three xenolines under hypoxia. In contrast, wild-type HSV-1 equally infected xenoline cells in normoxia and hypoxia, suggesting that the 34.5 mutation plays a role in the decreased C101 infectivity in hypoxia. Importantly, CD133(+) cells were not more resistant to oHSV than CD133(-) tumor cells regardless of oxygen tension. Furthermore, CD133 expression decreased as viral dose increased in two of the xenolines suggesting that up-regulation of CD133 in hypoxia was not the cause of reduced viral efficacy. Our findings that oHSV infectivity and cytotoxicity were diminished under hypoxia in several GBM xenolines likely have important implications for clinical applications of oHSV therapies, especially considering the vital role of hypoxia in the microenvironment of GBM tumors.     

Guanidylation and tail effects in cationic antimicrobial lipopeptoids

Background

Cationic antimicrobial peptides (CAMPs) are attractive scaffolds for the next generation of antimicrobial compounds, due to their broad spectrum of activity against multi-drug resistant bacteria and the reduced fitness of CAMP-insensitive mutants. Unfortunately, they are limited by poor in vivo performance, including ready cleavage by endogenous serum proteases.

Methodology/Principal Findings

To explore the potential for peptoid residues to replace well studied CAMP scaffolds we have produced a series of antimicrobial lipopeptoids, with sequences similar to previously reported lipopeptides. The activity of the peptoids was assessed against a panel of clinically relevant and laboratory reference bacteria, and the potential for non-specific binding was determined through hemolytic testing and repeating the antimicrobial testing in the presence of added bovine serum albumin (BSA). The most active peptoids displayed good to moderate activity against most of the Gram positive strains tested and moderate to limited activity against the Gram negatives. Antimicrobial activity was positively correlated with toxicity towards eukaryotic cells, but was almost completely eliminated by adding BSA.

Conclusion/Significance

The lipopeptoids had similar activities to the previously reported lipopeptides, confirming their potential to act as replacement, proteolytically stable scaffolds for CAMPs.     

Deciphering structural elements of mucin glycoprotein recognition

Mucin glycoproteins present a complex structural landscape arising from the multiplicity of glycosylation patterns afforded by their numerous serine and threonine glycosylation sites, often in clusters, and with variations in respective glycans. To explore the structural complexities in such glycoconjugates, we used NMR to systematically analyze the conformational effects of glycosylation density within a cluster of sites. This allows correlation with molecular recognition through analysis of interactions between these and other glycopeptides, with antibodies, lectins, and sera, using a glycopeptide microarray. Selective antibody interactions with discrete conformational elements, reflecting aspects of the peptide and disposition of GalNAc residues, are observed. Our results help bridge the gap between conformational properties and molecular recognition of these molecules, with implications for their physiological roles. Features of the native mucin motifs impact their relative immunogenicity and are accurately encoded in the antibody binding site, with the conformational integrity being preserved in isolated glycopeptides, as reflected in the antibody binding profile to array components.