A cluster of fourteen genes from enteropathogenic Escherichia coli is sufficient for the biogenesis of a type IV pilus

Enteropathogenic Escherichia coli (EPEC) adhere to epithelial cells in microcolonies, a pattern termed localized adherence (LA). LA is dependent upon the presence of 50–70MDa plasmids, termed EPEC adherence factor (EAF) plasmids. Expression of an EAF plasm id-encoded type IV fimbria, the bundle-forming pilus (BFP), is associated with the LA phenotype. TnphoA insertions in bfpA, the gene encoding the major structural subunit of the BFP, abolish LA. While bfpA::TnphoA mutants cannot be complemented for LA by plasmids carrying the bfpA gene alone in trans, this work shows that they can be complemented by plasmids carrying the bfpA gene, as well as approximately 10kb of downstream sequence, suggesting that such mutations have polar effects on downstream genes. The identification and characterization of a cluster of 13 genes immediately downstream of bfpA are described. The introduction into a laboratory Escherichia coli strain of a plasmid containing these 14 bfp gene cluster genes, along with pJPN14, a plasmid containing another fragment derived from the EAF plasmid, confers LA ability and BFP biogenesis. However, when a mutation is introduced into the last gene of the bfp cluster, neither LA nor BFP biogenesis is conferred. This work also provides evidence to show that the fragment cloned in pJPN14 encodes a factor(s) which results in increased levels of the pilin protein. Finally, it is shown that expression of the 14 genes in the bfp cluster from an IPTG-inducible promoter, in the absence of pJPN14, is sufficient to reconstitute BFP biogenesis in a laboratory E. cob strain, but is insufficient for LA. This is the first report demonstrating the reconstitution of a type IV pilus in a laboratory E. coli strain with a defined set of genes. The 8FP system should prove to be a useful model for studying the molecular mechanisms of type IV pilus biogenesis.     

Identification,Discrimination, and Discovery of Species of Marine Planktonic Ostracods Using DNA Barcodes

The Ostracoda (Crustacea; Class Ostracoda) is a diverse, frequently abundant, and ecologically important component of the marine zooplankton assemblage. There are more than 200 described species of marine planktonic ostracods, many of which (especially conspecific species) can be identified only by microscopic examination and dissection of fragile morphological characters. Given the complexity of species identification and increasing lack of expert taxonomists, DNA barcodes (short DNA sequences for species discrimination and identification) are particularly useful and necessary. Results are reported from analysis of 210 specimens of 78 species of marine planktonic ostracods, including two novel species, and 51 species for which barcodes have not been previously published. Specimens were collected during 2006 to 2008 from the Atlantic, Indian, and Southern Oceans, Greenland Sea and Gulf of Alaska. Samples were collected from surface to 5,000 m using various collection devices. DNA sequence variation was analyzed for a 598 base-pair region of the mitochondrial cytochrome oxidase subunit I (COI) gene. Kimura-2-Parameter (K2P) genetic distances within described species (mean = 0.010 ± 0.017 SD) were significantly smaller than between species (0.260 + 0.080), excluding eight taxa hypothesized to comprise cryptic species due to morphological variation (especially different size forms) and/or collection from different geographic regions. These taxa showed similar K2P distance values within (0.014 + 0.026) and between (0.221 ± 0.068) species. All K2P distances > 0.1 resulted from comparisons between identified or cryptic species, with no overlap between intra- and interspecific genetic distances. A Neighbor Joining tree resolved nearly all described species analyzed, with multiple sequences forming monophyletic clusters with high bootstrap values (typically 99%). Based on taxonomically and geographically extensive sampling and analysis (albeit with small sample sizes), the COI barcode region was shown to be a valuable character for discrimination, recognition, identification, and discovery of species of marine planktonic ostracods.     

Quantifying marine snow as a food choice for zooplankton using stable silicon isotope tracers

Aggregates of biogenic origin >0.5 mm, known as marine snow,represent a concentrated potential source of food for zooplankton.Little is known, however, about whether aggregates are commonlygrazed by zooplankton in the field. While previous laboratorystudies have shown that the euphausiid Euphausia pacifica, andthe copepod, Calanus pacificus, common crustacean zooplankters,consume marine snow if it is the only food source available,it is not known if euphausiids will select marine snow in thepresence of edible dispersed cells, as readily occurs in nature.To examine this question, we offered E. pacifica the diatom,Nitzschia angularis in aggregated and dispersed form as preysimultaneously. Aggregates and dispersed food contained identicalcell types so that differing prey quality, taste or motilitywould not be a factor. A new method was developed to track foodsources by labeling the frustules of aggregated cells with differentnaturally occurring, but rare, stable isotopes of silicon, ³⁰Siand ²⁹Si. Food selection was then estimated by measuring theisotopic composition of silica within fecal pellets producedby animals feeding on mixtures of the two labeled foods. Resultsindicate that E. pacifica consumed both aggregates and dispersedcells, even when more cells were made available in dispersedform than in aggregated form. This suggests that aggregatesmay indeed be a food source in the field, even when dispersedcells are relatively abundant. The method of labeling diatomcells with stable isotopes of silica may prove useful for futuregrazing experiments to distinguish identical cell types.     

Changing nuclear landscape and unique PML structures during early epigenetic transitions of human embryonic stem cells

The complex nuclear structure of somatic cells is important to epigenomic regulation, yet little is known about nuclear organization of human embryonic stem cells (hESC). Here we surveyed several nuclear structures in pluripotent and transitioning hESC. Observations of centromeres, telomeres, SC35 speckles, Cajal Bodies, lamin A/C and emerin, nuclear shape and size demonstrate a very different “nuclear landscape” in hESC. This landscape is remodeled during a brief transitional window, concomitant with or just prior to differentiation onset. Notably, hESC initially contain abundant signal for spliceosome assembly factor, SC35, but lack discrete SC35 domains; these form as cells begin to specialize, likely reflecting cell‐type specific genomic organization. Concomitantly, nuclear size increases and shape changes as lamin A/C and emerin incorporate into the lamina. During this brief window, hESC exhibit dramatically different PML‐defined structures, which in somatic cells are linked to gene regulation and cancer. Unlike the numerous, spherical somatic PML bodies, hES cells often display ～1–3 large PML structures of two morphological types: long linear “rods” or elaborate “rosettes”, which lack substantial SUMO‐1, Daxx, and Sp100. These occur primarily between Day 0–2 of differentiation and become rare thereafter. PML rods may be “taut” between other structures, such as centromeres, but clearly show some relationship with the lamina, where PML often abuts or fills a “gap” in early lamin A/C staining. Findings demonstrate that pluripotent hES cells have a markedly different overall nuclear architecture, remodeling of which is linked to early epigenomic programming and involves formation of unique PML‐defined structures. J. Cell. Biochem. 107: 609–621, 2009. © 2009 Wiley‐Liss, Inc.     

Partial purification and characterization of a gelatin-specific protease from the culture media of human pulmonary alveolar macrophages

A gelatin-specific protease from the culture media of human pulmonary alveolar macrophages has been partial purified by gel filtration and characterized. The macrophages were obtained by bronchopulmonary lavage from the lungs of disease-free smoking volunteers. The gelatin-specific protease initially requires trypsin activation. After chromatographing the culture media on a Sephadex G-200 column, trypsin is no longer required for activation. The gelatin-specific protease reported here shares many properties of previously reported gelatinases. It is inhibited by EDTA, cysteine, dithiothreitol and serum. It is unaffected by other protease inhibitors: phenylmethylsulfonyl fluoride, tosyllysine chloromethyl ketone and p-chloromercuribenzoate. Of all substrates tested activity was observed only with gelatin. It was inactive toward collagen, elastin and methemoglobin. This enzyme may have a role in the digestion of collagen which has been cleaved by a mammalian collagenase.     

Glomerular Injury Induced by Hydrogen Peroxide: Modifying Influence of Ace Inhibitors

The sensitivity of isolated glomeruli from normotensive (Wistar-Kyoto, WKY) and spontaneously hypertensive (SHR) strains to oxidant stress was studied by determining the incidence of pyknosis, karyohexis and karyolysis after incubation with different concentrations of hydrogen peroxide (H2O2) (4.7 × 10^-9-10^-3 M). Even though the proportion of glomeruli containing nuclei that demonstrated these features increased progressively with increasing concentrations of H2O2, the number of severely damaged glomeruli was relatively small even at concentrations of 4.7 × 10^-3 M.

Examination of the surface epithelial cells of glomeruli using scanning electron microscopy revealed no evidence of disturbance of the macroscopic or podocyte structure or, of increased blebbing after H2O2-treatment. These data suggest damage to nuclei is an early result of ROS stress on glomeruli.

Preincubation of WKY glomeruli with captopril or lisinopril resulted in a significant drop in the proportion of WKY glomeruli demonstrating structural damage after oxidant stress. In contrast, preincubation of SHR glomeruli with lisinopril had no effect on oxidant-induced changes in the morphology of SHR glomeruli, whereas captopril effected a significant increase in the proportion of glomeruli demonstrating damage at all concentrations of H2O2.     

Phenogenetics of triploid intersexes in Drosophila melanogaster

Triploid intersexes homozygous for a mutant (msl-2) known to impede the hyperactivation of the X chromosome in diploid males differentiate into adults, sexually indistinguishable from their heterozygous sibs. A shift toward female sexual differentiation mediated by manipulating the rearing temperature is accompanied by an apparent increase in the level of an X-linked gene product. This unexpected result is rationalized in terms of differential lethality of individuals at the two extremities of the distribution of X-activity levels in intersexes raised at a particular temperature. No evidence of a mosaicism comparable to the sexual mosaicism exhibited could be found with respect to an X-linked gene product in triploid intersexes.