p53-induced DNA bending and twisting: p53 tetramer binds on the outer side of a DNA loop and increases DNA twisting

DNA binding activity of p53 is crucial for its tumor suppressor function. Our recent studies have shown that four molecules of the DNA binding domain of human p53 (p53DBD) bind the response elements with high cooperativity and bend the DNA. By using A-tract phasing experiments, we find significant differences between the bending and twisting of DNA by p53DBD and by full-length human wild-type (wt) p53. Our data show that four subunits of p53DBD bend the DNA by 32–36°, whereas wt p53 bends it by 51–57°. The directionality of bending is consistent with major groove bends at the two pentamer junctions in the consensus DNA response element. More sophisticated phasing analyses also demonstrate that p53DBD and wt p53 overtwist the DNA response element by ≈35° and ≈70°, respectively. These results are in accord with molecular modeling studies of the tetrameric complex. Within the constraints imposed by the protein subunits, the DNA can assume a range of conformations resulting from correlated changes in bend and twist angles such that the p53–DNA tetrameric complex is stabilized by DNA overtwisting and bending toward the major groove at the CATG tetramers. This bending is consistent with the inherent sequence-dependent anisotropy of the duplex. Overall, the four p53 moieties are placed laterally in a staggered array on the external side of the DNA loop and have numerous interprotein interactions that increase the stability and cooperativity of binding. The novel architecture of the p53 tetrameric complex has important functional implications including possible p53 interactions with chromatin.

Induction of autoantibodies to mouse CCR5 with recombinant papillomavirus particles

The vertebrate immune system has evolved to respond vigorously to microbial infection but to ignore self-antigens. Evidence has emerged that B cell responses to viruses are initiated by immune recognition of ordered arrays of antigen on the viral surface. To test whether autoantibodies against a self-antigen can be induced by placing it in a context that mimics the ordered surface of a viral particle, a peptide representing an extracellular loop of the mouse chemokine receptor CCR5 was incorporated into an immunodominant site of the bovine papillomavirus virus L1 coat protein, which self-assembles into virus-like particles. Mice inoculated with chimeric L1-CCR5 particles generated autoantibodies that bound to native mouse CCR5, inhibited binding of its ligand RANTES, and blocked HIV-1 infection of an indicator cell line expressing a human-mouse CCR5 chimera. These results suggest a general method for inducing autoantibodies against self-antigens, with diverse potential basic research and clinical applications.

Microbial iron transport via a siderophore shuttle: A membrane ion transport paradigm

A mechanism of ion transport across membranes is reported. Microbial transport of Fe3+ generally delivers iron, a growth-limiting nutrient, to cells via highly specific siderophore-mediated transport systems. In contrast, iron transport in the fresh water bacterium Aeromonas hydrophila is found to occur by means of an indiscriminant siderophore transport system composed of a single multifunctional receptor. It is shown that (i) the siderophore and Fe3+ enter the bacterium together, (ii) a ligand exchange step occurs in the course of the transport, and (iii) a redox process is not involved in iron exchange. To the best of our knowledge, there have been no other reports of a ligand exchange mechanism in bacterial iron transport. The ligand exchange step occurs at the cell surface and involves the exchange of iron from a ferric siderophore to an iron-free siderophore already bound to the receptor. This ligand exchange mechanism is also found in Escherichia coli and seems likely to be widely distributed among microorganisms.

Oocytes are a source of catecholamines in the primate ovary: Evidence for a cell–cell regulatory loop

Catecholamines, thought to derive from the extrinsic innervation of the ovary, participate in the regulation of ovarian development and mature gonadal function. Recently, intraovarian neurons containing tyrosine hydroxylase (TH), the rate-limiting enzyme in catecholamine biosynthesis, were described in the ovary of nonhuman primates. We now show that the primate ovary expresses both the genes encoding TH and dopamine β-hydroxylase (DBH), the key enzymes in norepinephrine (NE) biosynthesis. Ovarian neurons were identified as a site of TH and DBH gene expression, and surprisingly, oocytes were identified as an exclusive site of DBH synthesis. Oocytes contain neither TH mRNA nor protein, indicating that they are unable to synthesize dopamine (DA). They did, however, express a DA transporter gene identical to that found in human brain. The physiological relevance of this transporter system and DBH in oocytes was indicated by the ability of isolated oocytes to metabolize exogenous DA into NE. Isolated follicles containing oocytes—but not those from which the oocytes had been removed—responded to DA with an elevation in cAMP levels; this elevation was prevented by propranolol, a β-adrenoreceptor antagonist. The results suggest that oocytes and somatic cells are linked by a neuroendocrine loop consisting of NE synthesized in oocytes from actively transported DA and cAMP produced by somatic follicular cells in response to NE-induced β-adrenoreceptor activation.

A TSG101/MDM2 regulatory loop modulates MDM2 degradation and MDM2/p53 feedback control

The p53 tumor suppressor protein and the MDM2 oncoprotein form a feedback-control loop that up-regulates cellular MDM2 production, blocks p53 activity, and promotes p53 decay. tsg101 was discovered as a gene whose deficiency results in neoplastic transformation of NIH 3T3 cells and the ability to generate metastatic tumors in nude mice. Its protein product contains a domain, Ubc, characteristic of the catalytic domain of ubiquitin conjugase (E2) enzymes but lacking an active-site cysteine crucial for ubiquitin conjugase activity. Here we report that TSG101 participates with MDM2 in an autoregulatory loop that modulates the cellular levels of both proteins, and also of p53, by affecting protein decay. We show that the Ubc domain of TSG101 interferes with ubiquitination of MDM2, that TSG101 inhibits MDM2 decay and elevates its steady-state level, and that these events are associated with down-regulation of p53 protein. Conversely, pulse–chase and Western blot experiments in wild-type and mutant fibroblasts indicate that elevation of MDM2 by overexpression of wild-type p53, by amplification of the endogenous MDM2 gene, or by transfection of MDM2-expressing constructs promotes TSG101 loss, which we show occurs by 26S proteasome-dependent decay. Our results identify TSG101 as both a regulator of, and target of, MDM2/p53 circuitry.

A phylogenomic approach to microbial evolution

To study the origin and evolution of biochemical pathways in microorganisms, we have developed methods and software for automatic, large-scale reconstructions of phylogenetic relationships. We define the complete set of phylogenetic trees derived from the proteome of an organism as the phylome and introduce the term phylogenetic connection as a concept that describes the relative relationships between taxa in a tree. A query system has been incorporated into the system so as to allow searches for defined categories of trees within the phylome. As a complement, we have developed the pyphy system for visualising the results of complex queries on phylogenetic connections, genomic locations and functional assignments in a graphical format. Our phylogenomics approach, which links phylogenetic information to the flow of biochemical pathways within and among microbial species, has been used to examine more than 8000 phylogenetic trees from seven microbial genomes. The results have revealed a rich web of phylogenetic connections. However, the separation of Bacteria and Archaea into two separate domains remains robust.

Prediction of operons in microbial genomes

Operon structure is an important organization feature of bacterial genomes. Many sets of genes occur in the same order on multiple genomes; these conserved gene groupings represent candidate operons. This study describes a computational method to estimate the likelihood that such conserved gene sets form operons. The method was used to analyze 34 bacterial and archaeal genomes, and yielded more than 7600 pairs of genes that are highly likely (P ≥ 0.98) to belong to the same operon. The sensitivity of our method is 30–50% for the Escherichia coli genome. The predicted gene pairs are available from our World Wide Web site http://www.tigr.org/tigr-scripts/operons/operons.cgi.

The Comprehensive Microbial Resource

One challenge presented by large-scale genome sequencing efforts is effective display of uniform information to the scientific community. The Comprehensive Microbial Resource (CMR) contains robust annotation of all complete microbial genomes and allows for a wide variety of data retrievals. The bacterial information has been placed on the Web at http://www.tigr.org/CMR for retrieval using standard web browsing technology. Retrievals can be based on protein properties such as molecular weight or hydrophobicity, GC-content, functional role assignments and taxonomy. The CMR also has special web-based tools to allow data mining using pre-run homology searches, whole genome dot-plots, batch downloading and traversal across genomes using a variety of datatypes.

A novel microbial habitat in the mid-ocean ridge subseafloor

The subseafloor at the mid-ocean ridge is predicted to be an excellent microbial habitat, because there is abundant space, fluid flow, and geochemical energy in the porous, hydrothermally influenced oceanic crust. These characteristics also make it a good analog for potential subsurface extraterrestrial habitats. Subseafloor environments created by the mixing of hot hydrothermal fluids and seawater are predicted to be particularly energy-rich, and hyperthermophilic microorganisms that broadly reflect such predictions are ejected from these systems in low-temperature (≈15°C), basalt-hosted diffuse effluents. Seven hyperthermophilic heterotrophs isolated from low-temperature diffuse fluids exiting the basaltic crust in and near two hydrothermal vent fields on the Endeavour Segment, Juan de Fuca Ridge, were compared phylogenetically and physiologically to six similarly enriched hyperthermophiles from samples associated with seafloor metal sulfide structures. The 13 organisms fell into four distinct groups: one group of two organisms corresponding to the genus Pyrococcus and three groups corresponding to the genus Thermococcus. Of these three groups, one was composed solely of sulfide-derived organisms, and the other two related groups were composed of subseafloor organisms. There was no evidence of restricted exchange of organisms between sulfide and subseafloor habitats, and therefore this phylogenetic distinction indicates a selective force operating between the two habitats. Hypotheses regarding the habitat differences were generated through comparison of the physiology of the two groups of hyperthermophiles; some potential differences between these habitats include fluid flow stability, metal ion concentrations, and sources of complex organic matter.

Solution structure of the A loop of 23S ribosomal RNA

The A loop is an essential RNA component of the ribosome peptidyltransferase center that directly interacts with aminoacyl (A)-site tRNA. The A loop is highly conserved and contains a ubiquitous 2′-O-methyl ribose modification at position U2552. Here, we present the solution structure of a modified and unmodified A-loop RNA to define both the A-loop fold and the structural impact of the U2552 modification. Solution data reveal that the A-loop RNA has a compact structure that includes a noncanonical base pair between C2556 and U2552. NMR evidence is presented that the N3 position of C2556 has a shifted pKa and that protonation at C2556-N3 changes the C-U pair geometry. Our data indicate that U2552 methylation modifies the A-loop fold, in particular the dynamics and position of residues C2556 and U2555. We compare our structural data with the structure of the A loop observed in a recent 50S crystal structure [Ban, N., Nissen, P., Hansen, J., Moore, P. B. & Steitz, T. A. (2000) Science 289, 905–920; Nissen, P., Hansen, J., Ban, N., Moore, P. B. & Steitz, T. A. (2000) Science 289, 920–930]. The solution and crystal structures of the A loop are dramatically different, suggesting that a structural rearrangement of the A loop must occur on docking into the peptidyltransferase center. Possible roles of this docking event, the shifted pKa of C2556 and the U2552 2′-O-methylation in the mechanism of translation, are discussed.

Microbial phyllosphere populations are more complex than previously realized

Phyllosphere microbial communities were evaluated on leaves of field-grown plant species by culture-dependent and -independent methods. Denaturing gradient gel electrophoresis (DGGE) with 16S rDNA primers generally indicated that microbial community structures were similar on different individuals of the same plant species, but unique on different plant species. Phyllosphere bacteria were identified from Citrus sinesis (cv. Valencia) by using DGGE analysis followed by cloning and sequencing of the dominant rDNA bands. Of the 17 unique sequences obtained, database queries showed only four strains that had been described previously as phyllosphere bacteria. Five of the 17 sequences had 16S similarities lower than 90% to database entries, suggesting that they represent previously undescribed species. In addition, three fungal species were also identified. Very different 16S rDNA DGGE banding profiles were obtained when replicate cv. Valencia leaf samples were cultured in BIOLOG EcoPlates for 4.5 days. All of these rDNA sequences had 97–100% similarity to those of known phyllosphere bacteria, but only two of them matched those identified by the culture independent DGGE analysis. Like other studied ecosystems, microbial phyllosphere communities therefore are more complex than previously thought, based on conventional culture-based methods.

Non-Arrhenius kinetics for the loop closure of a DNA hairpin

Intramolecular chain diffusion is an elementary process in the conformational fluctuations of the DNA hairpin-loop. We have studied the temperature and viscosity dependence of a model DNA hairpin-loop by FRET (fluorescence resonance energy transfer) fluctuation spectroscopy (FRETfs). Apparent thermodynamic parameters were obtained by analyzing the correlation amplitude through a two-state model and are consistent with steady-state fluorescence measurements. The kinetics of closing the loop show non-Arrhenius behavior, in agreement with theoretical prediction and other experimental measurements on peptide folding. The fluctuation rates show a fractional power dependence (β = 0.83) on the solution viscosity. A much slower intrachain diffusion coefficient in comparison to that of polypeptides was derived based on the first passage time theory of SSS [Szabo, A., Schulten, K. & Schulten, Z. (1980) J. Chem. Phys. 72, 4350–4357], suggesting that intrachain interactions, especially stacking interaction in the loop, might increase the roughness of the free energy surface of the DNA hairpin-loop.

Excitation–contraction coupling is unaffected by drastic alteration of the sequence surrounding residues L720–L764 of the α1S II-III loop

The II-III loop of the skeletal muscle dihydropyridine receptor (DHPR) α1S subunit is responsible for bidirectional-signaling interactions with the ryanodine receptor (RyR1): transmitting an orthograde, excitation–contraction (EC) coupling signal to RyR1 and receiving a retrograde, current-enhancing signal from RyR1. Previously, several reports argued for the importance of two distinct regions of the skeletal II-III loop (residues R681–L690 and residues L720–Q765, respectively), claiming for each a key function in DHPR–RyR1 communication. To address whether residues 720–765 of the II-III loop are sufficient to enable skeletal-type (Ca2+ entry-independent) EC coupling and retrograde interaction with RyR1, we constructed a green fluorescent protein (GFP)-tagged chimera (GFP-SkLM) having rabbit skeletal (Sk) DHPR sequence except for a II-III loop (L) from the DHPR of the house fly, Musca domestica (M). The Musca II-III loop (75% dissimilarity to α1S) has no similarity to α1S in the regions R681–L690 and L720–Q765. GFP-SkLM expressed in dysgenic myotubes (which lack endogenous α1S subunits) was unable to restore EC coupling and displayed strongly reduced Ca2+ current densities despite normal surface expression levels and correct triad targeting (colocalization with RyR1). Introducing rabbit α1S residues L720–L764 into the Musca II-III loop of GFP-SkLM (substitution for Musca DHPR residues E724–T755) completely restored bidirectional coupling, indicating its dependence on α1S loop residues 720–764 but its independence from other regions of the loop. Thus, 45 α1S-residues embedded in a very dissimilar background are sufficient to restore bidirectional coupling, indicating that these residues may be a site of a protein–protein interaction required for bidirectional coupling.

Desert dust suppressing precipitation: A possible desertification feedback loop

The effect of desert dust on cloud properties and precipitation has so far been studied solely by using theoretical models, which predict that rainfall would be enhanced. Here we present observations showing the contrary; the effect of dust on cloud properties is to inhibit precipitation. Using satellite and aircraft observations we show that clouds forming within desert dust contain small droplets and produce little precipitation by drop coalescence. Measurement of the size distribution and the chemical analysis of individual Saharan dust particles collected in such a dust storm suggest a possible mechanism for the diminished rainfall. The detrimental impact of dust on rainfall is smaller than that caused by smoke from biomass burning or anthropogenic air pollution, but the large abundance of desert dust in the atmosphere renders it important. The reduction of precipitation from clouds affected by desert dust can cause drier soil, which in turn raises more dust, thus providing a possible feedback loop to further decrease precipitation. Furthermore, anthropogenic changes of land use exposing the topsoil can initiate such a desertification feedback process.

Default taxonomy: Ernst Mayr’s view of the microbial world

This perspective is a response to a taxonomic proposal by E. Mayr [“Two empires or three?” (1998) Proc. Natl. Acad. Sci. USA 95, 9720–9723]. Mayr has suggested that the now accepted classification of life into three primary domains, Archaea, Bacteria, and Eucarya—originally proposed by myself and others—be abandoned in favor of the earlier Prokaryote–Eukaryote classification. Although the matter appears a taxonomic quibble, it is not that simple. At issue here are differing views as to the nature of biological classification, which are underlain by differing views as to what biology is and will be—matters of concern to all biologists.