This review is dedicated to Jeff Schell, one of the founders of modern`Agrobiology', the genetic and molecular dissection of crown gall disease. Together with notable scientists at the University of Gent, Belgium, Jeff spearheaded the discovery of the Ti-plasmid. The elusive`tumor inducing principle' was uncloaked and provided impetus for an incredibly fruitful subsequent 25 years of analyses. Scientists all over the world were caught up in unraveling the underlying mechanisms of Agrobacterium-mediated gene transfer to plants, and along the way uncovered a movable feast of fundamental insights. Below we summarize a sampling of Agrobacterium's most recently recognized accomplishments.
SUMMARY The panoply of microorganisms and other species present in our environment influence human health and disease, especially in cities, but have not been profiled with metagenomics at a city-wide scale. We sequenced DNA from surfaces across the entire New York City (NYC) subway system, the Gowanus Canal, and public parks. Nearly half of the DNA (48%) does not match any known organism; identified organisms spanned 1,688 bacterial, viral, archaeal, and eukaryotic taxa, which were enriched for harmless genera associated with skin (e.g., Acinetobacter). Predicted ancestry of human DNA left on subway surfaces can recapitulate U.S. Census demographic data, and bacterial signatures can reveal a station’s history, such as marine-associated bacteria in a hurricane-flooded station. Some evidence of pathogens was found (Bacillus anthracis), but a lack of reported cases in NYC suggests that the pathogens represent a normal, urban microbiome. This baseline metagenomic map of NYC could help long-term disease surveillance, bioterrorism threat mitigation, and health management in the built environment of cities.
Figure 3B has been corrected to show the general coverage of the Yersinia pestis pMT1 plasmid, but not the murine toxin gene (yMT). The initial claim of ''.consistent 203 coverage across the murine toxin gene.'' was erroneously based on looking at gene annotation coordinates from different reference sequences. No reads mapped to the yMT gene when updated annotations were used. The Summary, Results, and Discussion sections have been revised to remove and clarify misleading and speculative text about pathogenic organisms. We now state that although all our metagenomic analysis tools identified reads with similarity to B. anthracis and Y. pestis sequences, there is minimal coverage to the backbone genome of these organisms, and there is no strong evidence to suggest these organisms are in fact present, and no evidence of pathogenicity. The figure and the text have been corrected online and in the print version.
In order to perform their physiologic functions, polarized epithelial cells must target ion transport proteins to the appropriate domains of their plasma membranes. Molecular signals responsible for polarized sorting have been identified for several membrane proteins which span the bilayer once. Most ion transport proteins are polytopic, however, and little is known of the signals responsible for the targeting of this class of polypeptides. Members of the ␥-aminobutyric acid (GABA) transporter family are polytopic membrane proteins found endogenously in both epithelial cells and neurons. We have identified narrowly defined sequences which are required for the proper accumulation of two members of this transporter family in Madin-Darby canine kidney cells. The highly homologous GABA transporter isoforms, GAT-2 and GAT-3, localize to the basolateral and apical surfaces, respectively, when expressed stably in Madin-Darby canine kidney cells. We have generated deletion constructs and chimeric transporters composed of complimentary portions of GAT-2 and GAT-3. We find that information which directs their differential sorting is present in the C-terminal cytoplasmic tails of these two polypeptides. A sequence of 22 amino acids at the C terminus of GAT-2 is required for the transporter's basolateral distribution and is capable of directing GAT-3 to the basolateral surface when appended to the C terminus of this normally apical polypeptide. The deletion of 32 amino acids from the C terminus of GAT-3 causes this transporter to become mislocalized to both surfaces. Moreover, removal of the final three amino acids of GAT-3 (THF) similarly disrupts its apical sorting. The GAT-3 C-terminal sequence resembles motifs which interact with PDZ domains, raising the possibility that the steady state distribution of GAT-3 at the apical plasmalemmal surface requires a protein-protein interaction mediated by its extreme C-terminal cytoplasmic tail. These data provide the first characterization of a protein-based signal required for the apical distribution of a membrane protein.
In order to carry out their physiological functions, ion transport proteins must be targeted to the appropriate domains of cell membranes. Regulation of ion transport activity frequently involves the tightly controlled delivery of intracellular populations of transport proteins to the plasma membrane or the endocytic retrieval of transport proteins from the cell surface. Transport proteins carry signals embedded within their structures that specify their subcellular distributions and endow them with the capacity to participate in regulated membrane trafficking processes. Recently, a great deal has been learned about the biochemical nature of these signals, as well as about the cellular machinery that interprets them and acts upon their messages.
A series of new heterometallic gold(I) thiolates containing ferrocenyl-phoshines were synthesized. Their antimicrobial properties were studied and compared to that of FDA-approved drug, auranofin (Ridaura), prescribed for the treatment of rheumatoid arthritis. MIC in the order of one digit micromolar were found for most of the compounds against Gram-positive bacteria S. aureus and CA MRSA strains US300 and US400. Remarkably, auranofin inhibited S. aureus, US300 and US400 in the order of 150–300 nM. This is the first time that the potent inhibitory effect of auranofin on MRSA strains has been described. The effects of a selected heterometallic compound and auranofin were also studied in a non-tumorigenic human embryonic kidney cell line (HEK-293).
At least three high affinity Na+- and Cl--dependent gamma-aminobutyric acid (GABA) transporters are known to exist in the rat and mouse brain. These transporters share 50-65% amino acid sequence identity with the kidney betaine transporter which also transports GABA but with lower affinity. The betaine transporter (BGT) is expressed on the basolateral surface of polarized Madin-Darby canine kidney (MDCK) cells. Recent evidence suggests that the signals and mechanisms involved in membrane protein sorting share many functional characteristics in polarized neurons and epithelial cells. It was previously shown that the rat GABA transporter GAT-1 is located in the presynaptic membrane of axons where it plays a role in terminating GABAergic neurotransmission. When expressed in MDCK cells by transfection, GAT-1 was sorted to the apical membrane. In this report, we have localized the other two GABA transporters, GAT-2 and GAT-3, in transfected MDCK cells by GABA uptake, immunofluorescence, and cell surface biotinylation. GAT-3, like GAT-1, localized to the apical membrane of MDCK cells while GAT-2, like BGT, localized to the basolateral membrane. We have also expressed BGT in low density cultures of hippocampal neurons by microinjection and immunolocalized it to the dendrites. The distribution of GAT-3 in these neurons after transfection was axonal as well as somatodendritic. These results indicate that highly homologous subtypes of GABA transporters are sorted differently when expressed in epithelial cells or neurons and suggest that these two cell types share the capacity to distinguish among these isoforms and target them to distinct destinations.
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