H-NS controls metabolism and stress tolerance in Escherichia coli O157:H7 that influence mouse passage

Background  

H-NS is a DNA-binding protein with central roles in gene regulation and nucleoid structuring in Escherichia coli. There are over 60 genes that are influenced by H-NS many of which are involved in metabolism. To determine the significance of H-NS-regulated genes in metabolism and stress tolerance, an hns mutant of E. coli O157:H7 was generated (hns::nptI, FRIK47001P) and its growth, metabolism, and gastrointestinal passage compared to the parent strain (43895) and strain FRIK47001P harboring pSC0061 which contains a functional hns and 90-bp upstream of the open-reading frame.     

Contribution of dps to acid stress tolerance and oxidative stress tolerance in Escherichia coli O157:H7

An Escherichia coli O157:H7 dps::nptI mutant (FRIK 47991) was generated, and its survival was compared to that of the parent in HCl (synthetic gastric fluid, pH 1.8) and hydrogen peroxide (15 mM) challenges. The survival of the mutant in log phase (5-h culture) was significantly impaired (4-log(10)-CFU/ml reduction) compared to that of the parent strain (ca. 1.0-log(10)-CFU/ml reduction) after a standard 3-h acid challenge. Early-stationary-phase cells (12-h culture) of the mutant decreased by ca. 4 log(10) CFU/ml while the parent strain decreased by approximately 2 log(10) CFU/ml. No significant differences in the survival of late-stationary-phase cells (24-h culture) between the parent strain and the mutant were observed, although numbers of the parent strain declined less in the initial 1 h of acid challenge. FRIK 47991 was more sensitive to hydrogen peroxide challenge than was the parent strain, although survival improved in stationary phase. Complementation of the mutant with a functional dps gene restored acid and hydrogen peroxide tolerance to levels equal to or greater than those exhibited by the parent strain. These results demonstrate that decreases in survival were from the absence of Dps or a protein regulated by Dps. The results from this study establish that Dps contributes to acid tolerance in E. coli O157:H7 and confirm the importance of Dps in oxidative stress protection.     

Phylogeny and Phytogeography of Eupatorium (Eupatorieae, Asteraceae): Insights from Sequence Data of the nrDNA ITS Regions and cpDNA RFLP

Eupatorium were examined by sequencing the internal transcribed spacers (ITS) of nuclear ribosomal DNA and restriction site analysis of chloroplast DNA. Molecular data provided strong evidence that (1) this genus originated in North America, (2) the genus diverged into three morphological species groups, Eutrochium, Traganthes and Uncasia in North America, and (3) one of the North American Uncasia lineages migrated into temperate Europe and eastern Asia over the Bering land bridge. The estimated divergence times support a late Miocene to early Pliocene migration from North America to Eurasia via the Bering land bridge. A European species was sister to all of the eastern Asian species examined. The disjunct distribution pattern of the genus Eupatorium is incongruent with the classical Arcto-Tertiary geoflora concept. Received 13 September 1999/ Accepted in revised form 4 January 2000     

Chronic HIV Infection Enhances the Responsiveness of Antigen Presenting Cells to Commensal Lactobacillus

Chronic immune activation despite long-term therapy poses an obstacle to immune recovery in HIV infection. The role of antigen presenting cells (APCs) in chronic immune activation during HIV infection remains to be fully determined. APCs, the frontline of immune defense against pathogens, are capable of distinguishing between pathogens and non-pathogenic, commensal bacteria. We hypothesized that HIV infection induces dysfunction in APC immune recognition and response to some commensal bacteria and that this may promote chronic immune activation. Therefore we examined APC inflammatory cytokine responses to commensal lactobacilli. We found that APCs from HIV-infected patients produced an enhanced inflammatory response to Lactobacillus plantarum WCFS1 as compared to APCs from healthy, HIV-negative controls. Increased APC expression of TLR2 and CD36, signaling through p38-MAPK, and decreased expression of MAP kinase phosphatase-1 (MKP-1) in HIV infection was associated with this heightened immune response. Our findings suggest that chronic HIV infection enhances the responsiveness of APCs to commensal lactobacilli, a mechanism that may partly contribute to chronic immune activation.     

Knockdown, residual, and antifeedant activity of pyrethroids and home landscape bioinsecticides against Japanese beetles (Coleoptera: Scarabaeidae) on Linden foliage

Residual toxicity and leaf protection capability of five pyrethroids, professional and home garden azadirachtin formulations, and six other bioinsecticides for the home landscape were evaluated against the Japanese beetle, Popillia japonica Newman (Coleoptera: Scarabaeidae), on linden, Tilia cordata L. Capacity of intoxicated beetles to recover and subsequently feed and disperse also was evaluated to provide insight on activity characteristics of the different compounds. Intact shoots were sprayed and left in the field for varying intervals before being challenged with beetles in no-choice and choice tests. All pyrethroids except permethrin gave greater leaf protection, knockdown, and kill than did carbaryl, the standard, after 14 d of weathering. Deltamethrin, cyfluthrin, bifenthrin, and lamda-cyhalothrin gave a high level of protection for at least 19 d, and azadirachtin (Azatin XL) deterred feeding in choice tests for as long as 14 d. Home garden formulations containing pyrethrins in canola oil (Pyola) or azadiractin (Neem-Away) gave good short-term (< 3-d) protection. Formulations of capsaicin, rotenone + pyrethrins, kaolin particle film, D-limonene, or garlic extract were ineffective, the latter two formulations being highly phytotoxic to linden. Results of this study should help support updating of guidelines for insecticidal control of Japanese beetles.     

Molybdate treatment and sulfate starvation decrease ATP and DNA levels in Ferroplasma acidarmanus

Sulfate is a primary source of sulfur for most microbes and in some prokaryotes it is used an electron acceptor. The acidophile Ferroplasma acidarmanus (strain fer1) requires a minimum of 150 mM of a sulfate-containing salt for growth. Sulfate is assimilated by F. acidarmanus into proteins and reduced to form the volatile organic sulfur compounds methanethiol and dimethyldisulfide. In the absence of sulfate, cell death occurs by an unknown mechanism. In this study, cell viability and genomic DNA and ATP contents of F. acidarmanus were monitored in response to the absence of sulfate or the presence of sulfate and the sulfate analog molybdate ( MoO42- ). Cellular DNA and ATP contents were monitored as markers of cell viability. The absence of sulfate led to a decrease in viable cell numbers of greater than 7 log₁₀ within 5 days, a > 99% reduction in genomic DNA within 3 days, and a > 60% decrease in ATP within 6 h. Likewise, cells incubated with lost viability (decreased by > 2 log₁₀ in 5 days), extractable genomic DNA (reduction of > 60% in 2 days), and ATP (reduction of > 70 % in 2 hours). These results demonstrate that sulfate deprivation or the presence of molybdate have similar impacts on cell viability and essential biomolecules. Sulfate was coupled to cellular ATP content and maintenance of DNA integrity in F. acidarmanus, a finding that may be applicable to other acidophiles that are typically found in sulfate-rich biotopes.     

Contributions of the hippocampus and medial prefrontal cortex to energy and body weight regulation

The effects of selective ibotenate lesions of the complete hippocampus (CHip), the hippocampal ventral pole (VP), or the medial prefrontal cortex (mPFC) in male rats were assessed on several measures related to energy regulation (i.e., body weight gain, food intake, body adiposity, metabolic activity, general behavioral activity, conditioned appetitive responding). The testing conditions were designed to minimize the nonspecific debilitating effects of these surgeries on intake and body weight. Rats with CHip and VP lesions exhibited significantly greater weight gain and food intake compared with controls. Furthermore, CHip-lesioned rats, but not rats with VP lesions, showed elevated metabolic activity, general activity in the dark phase of the light-dark cycle, and greater conditioned appetitive behavior, compared with control rats without these brain lesions. In contrast, rats with mPFC lesions were not different from controls on any of these measures. These results indicate that hippocampal damage interferes with energy and body weight regulation, perhaps by disrupting higher-order learning and memory processes that contribute to the control of appetitive and consummatory behavior.     

Actin filaments elongate from their membrane-associated ends

In limulus sperm an actin filament bundle 55 mum in length extends from the acrosomal vacuole membrane through a canal in the nucleus and then coils in a regular fashion around the base of the nucleus. The bundle expands systematically from 15 filaments near the acrosomal vacuole to 85 filaments at the basal end. Thin sections of sperm fixed during stages in spermatid maturation reveal that the filament bundle begins to assemble on dense material attached to the acrosomal vacuole membrane. In micrographs fo these early stages in maturation, short bundles are seen extending posteriorly from the dense material. The significance is that these short, developing bundles have about 85 filaments, suggesting that the 85-filament end of the bundle is assembled first. By using filament bundles isolated and incubated in vitro with G actin from muscle, we can determine the end “preferred” for addition of actin monomers during polymerization. The end that would be associated with the acrosomal vacuole membrane, a membrane destined to be continuous with the plasma membrane, is preferred about 10 times over the other, thicker end. Decoration of the newly polymerized portions of the filament bundle with subfragment 1 of myosin reveals that the arrowheads point away from the acrosomal vacuole membrane, as is true of other actin filament bundles attached to membranes. From these observations we conclude that the bundle is nucleated from the dense material associated with the acrosomal vacuole and that monomers are added to the membrane-associated end. As monomers are added at the dense material, the thick first-made end of the filament bundle is pushed down through the nucleus where, upon reaching the base of the nucleus, it coils up. Tapering is brought about by the capping of the peripheral filaments in the bundle.     

Ultrastructural localization of amiloride-sensitive sodium channels and Na+,K(+)-ATPase in the rat's olfactory epithelial surface

Several studies have indicated that olfactory responses are impeded by amiloride. Therefore, it was of interest to see whether, and if so which, olfactory epithelial cellular compartments have amiloride- sensitive structures. Using ultrastructural methods that involved rapid freezing, freeze-substitution and low temperature embedding of olfactory epithelia, this study shows that, in the rat, this tissue is immunoreactive to antibodies against amiloride sensitive Na(+)- channels. However, microvilli of olfactory supporting cells, as opposed to receptor cilia, contained most of the immunoreactive sites. Apices from which the microvilli sprout and receptor cell dendritic knobs had much less if any of the amiloride-antibody binding sites. Using a direct ligand-binding cytochemical method, this study also confirms earlier ones that showed that olfactory receptor cell cilia have Na+, K(+)-ATPase. It is proposed that supporting cell microvilli and the receptor cilia themselves have mechanisms, different but likely complementary, that participate in regulating the salt concentration around the receptor cell cilia. In this way, both structures help to provide the ambient mucous environment for receptor cells to function properly. This regulation of the salt concentration of an ambient fluid environment is a function that the olfactory epithelium shares with cells of transporting epithelia, such as those of kidney.