Determination of the Stoichiometry between α- and γ1 Subunits of the BK Channel Using LRET

Two families of accessory proteins, β and γ, modulate BK channel gating and pharmacology. Notably, in the absence of internal Ca²⁺, the γ1 subunit promotes a large shift of the BK conductance-voltage curve to more negative potentials. However, very little is known about how α- and γ1 subunits interact. In particular, the association stoichiometry between both subunits is unknown. Here, we propose a method to answer this question using lanthanide resonance energy transfer. The method assumes that the kinetics of lanthanide resonance energy transfer-sensitized emission of the donor double-labeled α/γ1 complex is the linear combination of the kinetics of the sensitized emission in single-labeled complexes. We used a lanthanide binding tag engineered either into the α- or the γ1 subunits to bind Tb⁺³ as the donor. The acceptor (BODIPY) was attached to the BK pore-blocker iberiotoxin. We determined that γ1 associates with the α-subunit with a maximal 1:1 stoichiometry. This method could be applied to determine the stoichiometry of association between proteins within heteromultimeric complexes.     

Clostridium difficile heterogeneously impacts intestinal community architecture but drives stable metabolome responses

Clostridium difficile-associated diarrhoea (CDAD) is caused by C. difficile toxins A and B and represents a serious emerging health problem. Yet, its progression and functional consequences are unclear. We hypothesised that C. difficile can drive major measurable metabolic changes in the gut microbiota and that a relationship with the production or absence of toxins may be established. We tested this hypothesis by performing metabolic profiling on the gut microbiota of patients with C. difficile that produced (n=6) or did not produce (n=4) toxins and on non-colonised control patients (n=6), all of whom were experiencing diarrhoea. We report a statistically significant separation (P-value <0.05) among the three groups, regardless of patient characteristics, duration of the disease, antibiotic therapy and medical history. This classification is associated with differences in the production of distinct molecules with presumptive global importance in the gut environment, disease progression and inflammation. Moreover, although severe impaired metabolite production and biological deficits were associated with the carriage of C. difficile that did not produce toxins, only previously unrecognised selective features, namely, choline- and acetylputrescine-deficient gut environments, characterised the carriage of toxin-producing C. difficile. Additional results showed that the changes induced by C. difficile become marked at the highest level of the functional hierarchy, namely the metabolic activity exemplified by the gut microbial metabolome regardless of heterogeneities that commonly appear below the functional level (gut bacterial composition). We discuss possible explanations for this effect and suggest that the changes imposed by CDAD are much more defined and predictable than previously thought.     

A two-year report of pollen influx into Tauber traps in Mar Chiquita coastal lagoon,Buenos Aires,Argentina

Modern pollen deposition and its relationship to the surrounding vegetation were studied at a coastal lagoon from the southeast of Buenos Aires Province (Argentina). Tauber traps were monitored monthly over a 2-year period in a coastal dune barrier, salt marsh and continental freshwater lake. Pollen deposition exhibited seasonal patterns with maximum values during summer and a spatial variability of increasing deposition from the coast to inland sites. The pollen spectra suggest that airborne pollen originates mainly from local vegetation with scarce representation of extraregional sources. Herbaceous pollen predominates, comprising up to 90% of the total amount with Poaceae, Chenopodiineae and Asteroideae as the main types. Hydrophytic, psammophytic and extraregional types had little influence on the pollen spectra, generally comprising <5% of the total pollen. Pollen influx–vegetation abundance discrepancies were explained considering pollination syndrome, spatial distribution and structure of vegetation.     

Molecular Ecology of Listeria monocytogenes: Evidence for a Reservoir in Milking Equipment on a Dairy Farm

A longitudinal study aimed to detect Listeria monocytogenes on a New York State dairy farm was conducted between February 2004 and July 2007. Fecal samples were collected every 6 months from all lactating cows. Approximately 20 environmental samples were obtained every 3 months. Bulk tank milk samples and in-line milk filter samples were obtained weekly. Samples from milking equipment and the milking parlor environment were obtained in May 2007. Fifty-one of 715 fecal samples (7.1%) and 22 of 303 environmental samples (7.3%) were positive for L. monocytogenes. A total of 73 of 108 in-line milk filter samples (67.6%) and 34 of 172 bulk tank milk samples (19.7%) were positive for L. monocytogenes. Listeria monocytogenes was isolated from 6 of 40 (15%) sampling sites in the milking parlor and milking equipment. In-line milk filter samples had a greater proportion of L. monocytogenes than did bulk tank milk samples (P < 0.05) and samples from other sources (P < 0.05). The proportion of L. monocytogenes-positive samples was greater among bulk tank milk samples than among fecal or environmental samples (P < 0.05). Analysis of 60 isolates by pulsed-field gel electrophoresis (PFGE) yielded 23 PFGE types after digestion with AscI and ApaI endonucleases. Three PFGE types of L. monocytogenes were repeatedly found in longitudinally collected samples from bulk tank milk and in-line milk filters.Listeria monocytogenes can cause listeriosis in humans. This illness, despite being underreported, is an important public health concern in the United States (23) and worldwide. According to provisional incidence data provided by the Centers for Disease Control and Prevention (CDC), 762 cases of listeriosis were reported in the United States in 2007. In previous years (2003 to 2006), the number of reported annual listeriosis cases in the United States ranged between 696 and 896 cases per year (5).Exposure to food-borne L. monocytogenes may cause fever, muscle aches, and gastroenteritis (30), but does not usually cause septicemic illness in healthy nonpregnant individuals (7, 30). Elderly and immunocompromised people, however, are susceptible to listeriosis (22, 10), and they may develop more-severe symptoms (10). Listeriosis in pregnant women may cause abortion (22, 30) or neonatal death (22).Dairy products have been identified as the source of several human listeriosis outbreaks (4, 7, 10, 22). Listeria is ubiquitous on dairy farms (26), and it has been isolated from cows'' feces, feed (3, 26), and milk (21, 35). In ruminants, L. monocytogenes infections may be asymptomatic or clinical. Clinical cases typically present with encephalitis and uterine infections, often resulting in abortion (26, 39). Both clinically infected and healthy animals have been reported to excrete L. monocytogenes in their feces (20), which could eventually cause contamination of the bulk tank milk or milk-processing premises (39).On-farm epidemiologic research provides science-based information to improve farming and management practices. The Regional Dairy Quality Management Alliance (RDQMA) launched a combined United States Department of Agriculture (USDA)-RDQMA pilot project in January 2004 to scientifically validate intervention strategies in support of recommended best management practices among northeast dairy farms. The primary goal of the project was to track dynamics of infectious microorganisms on well-characterized dairy farms. Target species included Salmonella spp. (6, 36, 37), Mycobacterium avium subsp. paratuberculosis (13, 24), and L. monocytogenes.The objectives of this study were to describe the presence of L. monocytogenes on a dairy farm over time and to perform molecular subtyping by pulsed-field gel electrophoresis (PFGE) on L. monocytogenes isolates obtained from bulk tank milk, milk filters, milking equipment, feces, and the environmental samples to identify diversity among L. monocytogenes strains, persistence, and potential sources of bulk tank milk contamination.     

Altered kinetic properties of tyrosine-183 to cysteine mutation in glutamine synthetase of anabaena variabilis strain SA1 is responsible for excretion of ammonium ion produced by nitrogenase

A L-methionine- D, L-sulfoximine-resistant mutant of the cyanobacterium Anabaena variabilis, strain SA1, excreted the ammonium ion generated from N(2) reduction. In order to determine the biochemical basis for the NH(4)(+)-excretion phenotype, glutamine synthetase (GS) was purified from both the parent strain SA0 and from the mutant. GS from strain SA0 (SA0-GS) had a pH optimum of 7.5, while the pH optimum for GS from strain SA1 (SA1-GS) was 6.8. SA1-GS required Mn(+2) for optimum activity, while SA0-GS was Mg(+2) dependent. SA0-GS had the following apparent K(m) values at pH 7.5: glutamate, 1.7 m M; NH(4)(+), 0.015 m M; ATP, 0.13 m M. The apparent K(m) for substrates was significantly higher for SA1-GS at its optimum pH (glutamate, 9.2 m M; NH(4)(+), 12.4 m M; ATP, 0.17 m M). The amino acids alanine, aspartate, cystine, glycine, and serine inhibited SA1-GS less severely than the SA0-GS. The nucleotide sequences of glnA (encoding glutamine synthetase) from strains SA0 and SA1 were identical except for a single nucleotide substitution that resulted in a Y183C mutation in SA1-GS. The kinetic properties of SA1-GS isolated from E. coli or Klebsiella oxytoca glnA mutants carrying the A. variabilis SA1 glnA gene were also similar to SA1-GS isolated from A. variabilis strain SA1. These results show that the NH(4)(+)-excretion phenotype of A. variabilis strain SA1 is a direct consequence of structural changes in SA1-GS induced by the Y183C mutation, which elevated the K(m) values for NH(4)(+) and glutamate, and thus limited the assimilation of NH(4)(+) generated by N(2) reduction. These properties and the altered divalent cation-mediated stability of A. variabilis SA1-GS demonstrate the importance of Y183 for NH(4)(+) binding and metal ion coordination.     

Why are the genomes of endosymbiotic bacteria so stable?

The comparative analysis of three strains of the endosymbiotic bacterium Buchnera aphidicola has revealed high genome stability associated with an almost complete absence of chromosomal rearrangements and horizontal gene transfer events during the past 150 million years. The loss of genes involved in DNA uptake and recombination in the initial stages of endosymbiosis probably underlies this stability. Gene loss, which was extensive during the initial steps of Buchnera evolution, has continued in the different Buchnera lineages since their divergence.     

Side-stepping secondary symbionts: widespread horizontal transfer across and beyond the Aphidoidea

To elucidate the co-evolutionary relationships between phloem-feeding insects and their secondary, or facultative, bacterial symbionts, we explore the distributions of three such microbes--provisionally named the R-type (or PASS, or S-sym), T-type (or PABS), and U-type--across a number of aphid and psyllid hosts through the use of diagnostic molecular screening techniques and DNA sequencing. Although typically maternally transmitted, phylogenetic and pairwise divergence analyses reveal that these bacteria have been independently acquired by a variety of unrelated insect hosts, indicating that horizontal transfer has helped to shape their distributions. Based on the high genetic similarity between symbionts in different hosts, we argue that transfer events have occurred recently on an evolutionary timescale. In several instances, however, closely related symbionts associate with related hosts, suggesting that horizontal transfer between distant relatives may be rarer than transmission between close relatives. Our findings on the prevalence of these symbionts within many aphid taxa, along with published observations concerning their effects on host fitness, imply a significant role of facultative symbiosis in aphid ecology and evolution.     

Phlebotomine sand flies in Porteirinha, an area of American visceral leishmaniasis transmission in the State of Minas Gerais, Brazil

A study of the phlebotomine sand fly fauna was carried out in an endemic area of American visceral leishmaniasis (AVL) in the municipality of Porteirinha, in the Brazilian state of Minas Gerais. Captures were performed with CDC light traps in 7 districts, 5 days per month, during 2 consecutive years (January 2000 to December 2001). A total of 3240 sand flies were captured and identified. Sixteen species were found, among which 15 belonged to the genus Lutzomyia and one to the genus Brumptomyia. Lutzomyia longipalpis, a proven vector of AVL, was the predominant species (71.85%) throughout the time period. The interference of climatic factors (temperature, humidity, and rainfall) over the populational dynamics of the sand flies was determined. Statistical analysis of the data showed a significant correlation among the number of phlebotomine sand flies collected, rainfall, and humidity, whereas the effect of temperature was negligible, in that particular region. The amount of collected phlebotomine, the number of human cases, and the prevalence of canine AVL in the districts of Porteirinha are discussed.