Multiple cholesterol recognition/interaction amino acid consensus (CRAC) motifs in cytosolic C tail of Slo1 subunit determine cholesterol sensitivity of Ca2+- and voltage-gated K+ (BK) channels

Large conductance, Ca(2+)- and voltage-gated K(+) (BK) channel proteins are ubiquitously expressed in cell membranes and control a wide variety of biological processes. Membrane cholesterol regulates the activity of membrane-associated proteins, including BK channels. Cholesterol modulation of BK channels alters action potential firing, colonic ion transport, smooth muscle contractility, endothelial function, and the channel alcohol response. The structural bases underlying cholesterol-BK channel interaction are unknown. Such interaction is determined by strict chemical requirements for the sterol molecule, suggesting cholesterol recognition by a protein surface. Here, we demonstrate that cholesterol action on BK channel-forming Cbv1 proteins is mediated by their cytosolic C tail domain, where we identified seven cholesterol recognition/interaction amino acid consensus motifs (CRAC4 to 10), a distinct feature of BK proteins. Cholesterol sensitivity is provided by the membrane-adjacent CRAC4, where Val-444, Tyr-450, and Lys-453 are required for cholesterol sensing, with hydrogen bonding and hydrophobic interactions participating in cholesterol location and recognition. However, cumulative truncations or Tyr-to-Phe substitutions in CRAC5 to 10 progressively blunt cholesterol sensitivity, documenting involvement of multiple CRACs in cholesterol-BK channel interaction. In conclusion, our study provides for the first time the structural bases of BK channel cholesterol sensitivity; the presence of membrane-adjacent CRAC4 and the long cytosolic C tail domain with several other CRAC motifs, which are not found in other members of the TM6 superfamily of ion channels, very likely explains the unique cholesterol sensitivity of BK channels.     

Host-microbe interactions that shape the pathogenesis of Acinetobacter baumannii infection

Acinetobacter baumannii is an opportunistic pathogen that has emerged as a prevalent source of nosocomial infections, most frequently causing ventilator‐associated pneumonia. The emergence of pan‐drug resistant strains magnifies the problem by reducing viable treatment options and effectively increasing the mortality rate associated with Acinetobacter infections. In light of this rising threat, research on A. baumannii epidemiology, antibiotic resistance, and pathogenesis is accelerating. The recent development of both in vitro and in vivo models has enabled studies probing the host–Acinetobacter interface. Bacterial genetic screens and comparative genomic studies have led to the identification of several A. baumannii virulence factors. Additionally, investigations into host defence mechanisms using animal models or cell culture have provided insight into the innate immune response to infection. This review highlights some of the key attributes of A. baumannii virulence with an emphasis on bacterial interactions with the innate immune system.     

Oxidative damage to DNA related to survivorship and carotenoid-based sexual ornamentation in the common yellowthroat

Carotenoid-based sexual ornaments are hypothesized to be reliable signals of male quality, based on an allocation trade-off between the use of carotenoids as pigments and their use in antioxidant defence against reactive oxygen species. Carotenoids appear to be poor antioxidants in vivo, however, and it is not clear whether variation in ornament expression is correlated with measures of oxidative stress (OXS) under natural conditions. We used single-cell gel electrophoresis to assay oxidative damage to erythrocyte DNA in the common yellowthroat (Geothlypis trichas), a sexually dichromatic warbler in which sexual selection favours components of the males' yellow 'bib'. We found that the level of DNA damage sustained by males predicted their overwinter survivorship and was reflected in the quality of their plumage. Males with brighter yellow bibs showed lower levels of DNA damage, both during the year the plumage was sampled (such that yellow brightness signalled current OXS) and during the previous year (such that yellow brightness signalled past OXS). We suggest that carotenoid-based ornaments can convey information about OXS to prospective mates and that further work exploring the proximate mechanism(s) linking OXS to coloration is warranted.     

GxG epistasis in growth and condition and the maintenance of genetic polymorphism in Gambusia holbrooki

Theory on indirect genetic effects (IGEs) indicates that variation in the genetic composition of social groups can generate GxG epistasis that may promote the evolution of stable polymorphisms. Using a livebearing fish with a genetic polymorphism in coloration and associated behavioral differences, we tested whether genotypes of social partners interacted with focal individual genotypes to influence growth and condition over 16 weeks of development. We found that IGEs had a significant influence on patterns of feeding, regardless of focal fish genotype. There was no influence of social environment on juvenile length, but there was significant GxG epistasis for body condition. Each focal juvenile was in better condition when its own genotype was not present in adult social partners. These data are consistent with negative frequency‐dependent selection in which each morph performs better when it is rare. Neither variation in feeding nor activity‐related behaviors explained variation in body condition, suggesting that GxG epistasis for condition was caused by physiological differences between the two genotypes. These findings indicate that GxG epistasis in a given polymorphism can generate fitness landscapes that contribute to the maintenance of that polymorphism and to maintenance of genetic variation for additional fitness‐related traits.     

Integrated Recombinant Protein Expression and Purification Platform Based on Ralstonia eutropha

Protein purification of recombinant proteins constitutes a significant cost of biomanufacturing and various efforts have been directed at developing more efficient purification methods. We describe a protein purification scheme wherein Ralstonia eutropha is used to produce its own “affinity matrix,” thereby eliminating the need for external chromatographic purification steps. This approach is based on the specific interaction of phasin proteins with granules of the intracellular polymer polyhydroxybutyrate (PHB). By creating in-frame fusions of phasins and green fluorescent protein (GFP) as a model protein, we demonstrated that GFP can be efficiently sequestered to the surface of PHB granules. In a second step, we generated a phasin-intein-GFP fusion, wherein the self-cleaving intein can be activated by the addition of thiols. This construct allowed for the controlled binding and release of essentially pure GFP in a single separation step. Finally, pure, active β-galactosidase was obtained in a single step using the above described method.     

Reproduction of Meloidogyne incognita on Winter Cover Crops Used in Cotton Production

Substantial reproduction of Meloidogyne incognita on winter cover crops may lead to damaging populations in a subsequent cotton (Gossypium hirsutum) crop. The amount of population increase during the winter depends on soil temperature and the host status of the cover crop. Our objectives were to quantify M. incognita race 3 reproduction on rye (Secale cereale) and several leguminous cover crops and to determine if these cover crops increase population densities of M. incognita and subsequent damage to cotton. The cover crops tested were ‘Bigbee’ berseem clover (Trifolium alexandrinum), ‘Paradana’ balansa clover (T. balansae), ‘AU Sunrise’ and ‘Dixie’ crimson clover (T. incarnatum), ‘Cherokee’ red clover (T. pratense), common and ‘AU Early Cover’ hairy vetch (Vicia villosa), ‘Cahaba White’ vetch (V. sativa), and ‘Wrens Abruzzi’ rye. In the greenhouse tests, egg production was greatest on berseem clover, Dixie crimson clover, AU Early Cover hairy vetch, and common hairy vetch; intermediate on Balansa clover and AU Sunrise crimson clover; and least on rye, Cahaba White vetch, and Cherokee red clover. In both 2002 and 2003 field tests, enough heat units were accumulated between 1 January and 20 May for the nematode to complete two generations. Both AU Early Cover and common hairy vetch led to greater root galling than fallow in the subsequent cotton crop; they also supported high reproduction of M. incognita in the greenhouse. Rye and Cahaba White vetch did not increase root galling on cotton and were relatively poor hosts for M. incognita. Only those legumes that increased populations of M. incognita reduced cotton yield. In the southern US, M. incognita can complete one to two generations on a susceptible winter cover crop, so cover crops that support high nematode reproduction may lead to damage and yield losses in the following cotton crop. Planting rye or Meloidogyne-resistant legumes as winter cover crops will lower the risk of increased nematode populations compared to most vetches and clovers.