Evolutionary origins and sequence of the Escherichia coli O4 O-antigen gene cluster

Escherichia coli express many types of O antigen, present in the outer membrane of the Gram-negative bacterial cell wall. O-Antigen biosynthesis genes are clustered together and differences seen in O-antigen types are due to genetic variation within this gene cluster. Sequencing of the E. coli O4 O-antigen gene cluster revealed a similar gene order and high levels of similarity to that of E. coli O26; indicating a common ancestor. These lateral transfer events observed within O-antigen gene clusters may occur as part of the evolution of the pathogenic clones.     

ChemDB: a public database of small molecules and related chemoinformatics resources

MOTIVATION: The development of chemoinformatics has been hampered by the lack of large, publicly available, comprehensive repositories of molecules, in particular of small molecules. Small molecules play a fundamental role in organic chemistry and biology. They can be used as combinatorial building blocks for chemical synthesis, as molecular probes in chemical genomics and systems biology, and for the screening and discovery of new drugs and other useful compounds. RESULTS: We describe ChemDB, a public database of small molecules available on the Web. ChemDB is built using the digital catalogs of over a hundred vendors and other public sources and is annotated with information derived from these sources as well as from computational methods, such as predicted solubility and three-dimensional structure. It supports multiple molecular formats and is periodically updated, automatically whenever possible. The current version of the database contains approximately 4.1 million commercially available compounds and 8.2 million counting isomers. The database includes a user-friendly graphical interface, chemical reactions capabilities, as well as unique search capabilities. AVAILABILITY: Database and datasets are available on http://cdb.ics.uci.edu.     

Inhibitors of the inducible microsomal prostaglandin E2 synthase (mPGES-1) derived from MK-886

A series of potent and selective inhibitors of the inducible microsomal PGE2 synthase (mPGES-1) has been developed based on the indole FLAP inhibitor MK-886. Compounds 23 and 30 inhibit mPGES-1 with potencies in the low nanomolar range and with selectivities of at least 100-fold compared to their inhibition of mPGES-2, thromboxane synthase and binding affinity to FLAP. They also block the production of PGE2 in cell based assays but with a decreased potency and more limited selectivity compared to the enzyme assays.     

Evidence for genetic drift in the diversification of a geographically isolated population of the hyperthermophilic archaeon Pyrococcus

Genetic drift is a mechanism of population divergence that is important in the evolution of plants and animals but is thought to be rare in free-living microorganisms because of their typically large population sizes and unrestricted means of dispersal. We used both phylogenetic and insertion sequence (IS) element analyses in hyperthermophilic archaea of the genus Pyrococcus to test the hypothesis that genetic drift played an important role in the diversification of these microorganisms. Multilocus sequence typing of a collection of 36 isolates of Pyrococcus, from different hydrothermal systems in the Pacific Ocean and the Mediterranean Sea, revealed that Pyrococcus populations from different geographic locations are genetically differentiated. Analysis of IS elements in these isolates exposed their presence in all individuals of only one geographically isolated lineage, that of Vulcano Island in the Mediterranean Sea. Detailed sequence analysis of six selected IS elements in the Vulcano population showed that these elements cause deleterious genomic alterations, including inactivation of gene function. The high frequency of IS elements in the sampled population together with their observed harmful effects in the genome of Pyrococcus provide molecular evidence that the Vulcano Island population of Pyrococcus is geographically isolated and that those genetic mobile elements have been brought up to high frequency by genetic drift. Thus, genetic drift resulting from physical isolation should be considered as a factor influencing differentiation in prokaryotes.     

Sequence of the Escherichia coli O26 O antigen gene cluster and identification of O26 specific genes

Escherichia coli associated with outbreaks of gastroenteritis and hemolytic uremic syndrome include clones with O antigens O157 and O111. However, O26 has emerged as an O antigen present in pathogenic strains, particularly those implicated in cases of infantile gastroenteritis worldwide. The O26 O antigen gene cluster was sequenced. It was found to contain the genes expected for biosynthesis of nucleotide sugars L-rhamnose, N-acetyl-L-fucosamine and N-acetyl-glucosamine, as well genes for O unit flippase, O antigen polymerase and potential transferase genes. By polymerase chain reaction testing against representative strains for the 166 Escherichia coli O serogroups and some randomly selected Gram-negative bacteria, we identified three O antigen genes that are highly specific to O26. This work provides the basis for a sensitive test for the rapid detection of pathogenic clones with the O26 antigen, which has implications for public health, especially in the control of food-borne outbreaks.     

Protein degradation in the rumen of red clover forage at various stages of growth and conserved as silage or wrapped big bales

In order to study the extent to which rumen soluble nitrogen can contribute to the intestinal flow, a study was carried out to simultaneously assess the dynamics of protein disappearance from dacron bags placed in the rumen and the amount of various N products in the rumen fluid (total nitrogen (tN), ammonia nitrogen (NH3-N), non-ammonia nitrogen (NAN)). The measurements were carried out on 4 sheep fed successively various red clover forages. These forages included the initial growth of fresh red clover (50% bud, first flower, and full flower). In addition, one silage and one wrapped big bale at the first flower stage and two wrapped big bales (harvested at 51% and 71% dry matter) at the full flower stage were given. The effective degradability of nitrogen (DegN) for a fresh forage estimated from the nylon bag procedure did not vary (p > 0.05) with the vegetation stage (0.727 for the bud stage, 0.694 at the first flower, 0.706 at the full flower). The DegN of the silage was higher (p < 0.05; 0.735) and the DegN of the wrapped big bale was markedly lower (p < 0.05; 0.660), than the original fresh forage at the first flower. The DegN of the wrapped big bales made at 51 and 71% DM, respectively, were 0.625 and 0.604 against 0.706 for fresh forage at the full flower stage. The concentrations of tN and NAN in the rumen fluid were low, highest 1 h to 2 h after feeding, and then decreasing up to 7 h after feeding whatever the growth stage and conservation mode. A part of the solubilised nitrogen remained as protein 1 h after feeding for fresh red clover harvested at various growth stages, while minimal protein could be seen in the rumen fluid after the sheep were fed silage or wrapped big bales. The part of NAN escaping rumen degradation and transiting with the rumen fluid was between 7 and 13% of the nitrogen disappearing from the nylon bags (NAN/CP x DegN) placed in the rumen. There was only a small difference for forages at different stages of growth, or modes of conservation. This fraction was higher for wrapped big bales and particularly for the late stage forage (wrapped big bale, 71% DM, harvested at the full flower stage).     

Rafts promote assembly and atypical targeting of a nonenveloped virus, rotavirus, in Caco-2 cells

Rotavirus follows an atypical pathway to the apical membrane of intestinal cells that bypasses the Golgi. The involvement of rafts in this process was explored here. VP4 is the most peripheral protein of the triple-layered structure of this nonenveloped virus. High proportions of VP4 associated with rafts within the cell as early as 3 h postinfection. In the meantime a significant part of VP4 was targeted to the Triton X-100-resistant microdomains of the apical membrane, suggesting that this protein possesses an autonomous signal for its targeting. At a later stage the other structural rotavirus proteins were also found in rafts within the cells together with NSP4, a nonstructural protein required for the final stage of virus assembly. Rafts purified from infected cells were shown to contain infectious particles. Finally purified VP4 and mature virus were shown to interact with cholesterol- and sphingolipid-enriched model lipid membranes that changed their phase preference from inverted hexagonal to lamellar structures. Together these results indicate that a direct interaction of VP4 with rafts promotes assembly and atypical targeting of rotavirus in intestinal cells.