Horizontal gene transfer of PIB-type ATPases among bacteria isolated from radionuclide- and metal-contaminated subsurface soils

Aerobic heterotrophs were isolated from subsurface soil samples obtained from the U.S. Department of Energy's (DOE) Field Research Center (FRC) located at Oak Ridge, Tenn. The FRC represents a unique, extreme environment consisting of highly acidic soils with co-occurring heavy metals, radionuclides, and high nitrate concentrations. Four hundred isolates obtained from contaminated soil were assayed for heavy metal resistance, and a smaller subset was assayed for tolerance to uranium. The vast majority of the isolates were gram-positive bacteria and belonged to the high-G+C- and low-G+C-content genera Arthrobacter and Bacillus, respectively. Genomic DNA from a randomly chosen subset of 50 Pb-resistant (Pb(r)) isolates was amplified with PCR primers specific for P(IB)-type ATPases (i.e., pbrA/cadA/zntA). A total of 10 pbrA/cadA/zntA loci exhibited evidence of acquisition by horizontal gene transfer. A remarkable dissemination of the horizontally acquired P(IB)-type ATPases was supported by unusual DNA base compositions and phylogenetic incongruence. Numerous Pb(r) P(IB)-type ATPase-positive FRC isolates belonging to the genus Arthrobacter tolerated toxic concentrations of soluble U(VI) (UO(2)(2+)) at pH 4. These unrelated, yet synergistic, physiological traits observed in Arthrobacter isolates residing in the contaminated FRC subsurface may contribute to the survival of the organisms in such an extreme environment. This study is, to the best of our knowledge, the first study to report broad horizontal transfer of P(IB)-type ATPases in contaminated subsurface soils and is among the first studies to report uranium tolerance of aerobic heterotrophs obtained from the acidic subsurface at the DOE FRC.     

Purification of cysteine proteinases from trichomonads using bacitracin-sepharose

Abstract Bacitracin affinity chromatography has been used to purify proteinases of the parasitic protozoon Tritrichomonas foetus . It proved superior to other affinity chromatography methods we have tested for the purification of trichomonad proteinases and should prove a useful procedure for purifying cysteine proteines from these parasites and other parasitic protozoa. The main cysteine proteinases of T. foetus were purified over 100-fold to be free from the majority of other cell proteins. About 90 μg of protein containing 1.56-fold more proteinase activity than was detectable in the original cell lysate was obtained from 10⁹ cells (7.2 mg protein). SDS-PAGE revealed that the eluate contained two main Coomassie blue-staining bands. N-terminal amino acid sequence analysis of these proteins confirmed that one of them was a cysteine proteinase with unusuall features. Cysteine proteinases were also purified from cell lysates of Trichomonas vaginalis and a N-terminal sequence determined. This is the first amino acid sequence information that has been obtained for trichomonad cysteine proteinases. The method was also used to purify proteinases from the medium of T. foetus cultures. Some selectivity in binding of the proteinases to the affinity column was found.     

TLR2 Mediates Recognition of Live Staphylococcus epidermidis and Clearance of Bacteremia

Background

Staphylococcus epidermidis (SE) is a nosocomial pathogen that causes catheter-associated bacteremia in the immunocompromised, including those at the extremes of age, motivating study of host clearance mechanisms. SE-derived soluble components engage TLR2; but additional signaling pathways have also been implicated, and TLR2 can play complex, at times detrimental, roles in host defense against other Staphylococcal spp. The role of TLR2 in responses of primary blood leukocytes to live SE and in clearance of SE bacteremia, the most common clinical manifestation of SE infection, is unknown.

Methodology/Principal Findings

We studied TLR2-mediated recognition of live clinical SE strain 1457 employing TLR2-transfected cells, neutralizing anti-TLR antibodies and TLR2-deficient mice. TLR2 mediated SE-induced cytokine production in human embryonic kidney cells, human whole blood and murine primary macrophages, in part via recognition of a soluble TLR2 agonist. After i.v. challenge with SE, early (1 h) cytokine/chemokine production and subsequent clearance of bacteremia (24–48 h) were markedly impaired in TLR2-deficient mice.

Conclusions/Significance

TLR2 mediates recognition of live SE and clearance of SE bacteremia in vivo.     

Negative regulation of LRP6 function by casein kinase I epsilon phosphorylation

Wnt signaling acts in part through the low density lipoprotein receptor-related transmembrane proteins LRP5 and LRP6 to regulate embryonic development and stem cell proliferation. Up-regulated signaling is associated with many forms of cancer. Casein kinase I epsilon (CKIepsilon) is a known component of the Wnt-beta-catenin signaling pathway. We find that CKIepsilon binds to LRP5 and LRP6 in vitro and in vivo and identify three CKIepsilon-specific phosphorylation sites in LRP6. Two of the identified phosphorylation sites, Ser1420 and Ser1430, influence Wnt signaling in vivo, since LRP6 with mutation of these sites is a more potent activator of both beta-catenin accumulation and Lef-1 reporter activity. Whereas Wnt3a regulates CKIepsilon kinase activity, LRP6 does not, placing CKIepsilon upstream of LRP6. Mutation of LRP6 Ser1420 and Ser1430 to alanine strengthens its interaction with axin, suggesting a mechanism by which CKIepsilon may negatively regulate Wnt signaling. The role of CKIepsilon is therefore more complex than was previously appreciated. Generation of active CKIepsilon may induce a negative feedback loop by phosphorylation of sites on LRP5/6 that modulate axin binding and hence beta-catenin degradation.     

Functional Demonstration of Connexin—Protein Binding Using Surface Plasmon Resonance

Surface plasmon resonance (SPR) allows examination of protein-protein interactions in real time, from which both binding affinities and kinetics can be directly determined. We have used the SPR technique to search for proteins in heart tissue that would be candidate binding partners for the cardiac gap junction protein, connexin43 (Cx43). Heart lysate showed a strong, pH-dependent binding to the carboxyl terminus (CT) of Cx43 (amino acids 254-382) covalently linked to an SPR cuvette. Binding was inhibited by the presence of v-src transfected 3T3 cell lysate, suggesting that binding partners in these two lysates may compete for overlapping epitopes on Cx43CT. The combined application of proteomic and functional studies is expected to identify which proteins within heart tissue interact with Cx43 and what roles they may play in gap junction function.     

The amitochondriate eukaryote Trichomonas vaginalis contains a divergent thioredoxin-linked peroxiredoxin antioxidant system

Trichomonas is an amitochondriate parasitic protozoon specialized for an anaerobic lifestyle. Nevertheless, it is exposed to oxygen and is able to cope with the resultant oxidative stress. In the absence of glutathione, cysteine has been thought to be the major antioxidant. We now report that the parasite contains thioredoxin reductase, which functions together with thioredoxin and thioredoxin peroxidase to detoxify potentially damaging oxidants. Thioredoxin reductase and thioredoxin also reduce cystine and so may play a role in maintaining the cellular cysteine levels. The importance of the thioredoxin system as one of the major antioxidant defense mechanisms in Trichomonas was confirmed by showing that the parasite responds to environmental changes resulting in increased oxidative stress by up-regulating thioredoxin and thioredoxin peroxidases levels. Sequence data indicate that the thioredoxin reductase of Trichomonas differs fundamentally in structure from that of its human host and thus may represent a useful drug target. The protein is generally similar to thioredoxin reductases present in other lower eukaryotes, all of which probably originated through horizontal gene transfer from a prokaryote. The phylogenetic signal in thioredoxin peroxidase is weak, but evidence from trees suggests that this gene has been subject to repeated horizontal gene transfers from different prokaryotes to different eukaryotes. The data are thus consistent with the complexity hypothesis that predicts that the evolution of simple pathways such as the thioredoxin cascade are likely to be affected by horizontal gene transfer between species.