Reversion and immobilization of phage Mud1 cts (Apr lac) insertion mutations in Salmonella typhimurium

Summary Phage Mud1 cts (Ap^r lac), or Mud1, insertion mutations may be accompanied by adjacent deletion formation which can complicate use of lac fusions generated with this phage for gene regulatory studies. As for phage Mu insertion mutations, phage Mud1 insertions fail to revert at significant frequency (whether or not accompanied by an adjacent deletion). We describe isolation of revertible (X mutant) derivatives of phage Mud1 in Salmonella typhimurium. The X mutant derivatives allow use of reversion as a simple test to determine whether a Mud1 insertion has occurred precisely without an adjacent deletion that may have fused the lac genes to a promoter outside of the gene of interest. In addition, a simple method for stabilizing Mud1 generated lac fusions against subsequent transposition is described.     

Mutational analysis of the histidine operon promoter of Salmonella typhimurium.

We isolated a collection of 67 independent, spontaneous Salmonella typhimurium his operon promoter mutants with decreased his expression. The mutants were isolated by selecting for resistance to the toxic lactose analog o-nitrophenyl-beta-D-thiogalactoside in a his-lac fusion strain. The collection included base pair substitutions. small insertions, a deletion, and one large insertion identified as IS30 (IS121), which is resident on the Mu d1 cts(Apr lac) phage used to construct the his-lac fusion. Of the 37 mutations that were sequenced, 14 were unique. Six of the 14 were isolated more than once, with the IS30 insertion occurring 16 times. The mutations were located throughout the his promoter region, with two in the conserved - 35 hexamer sequence, four in the conserved - 10 hexamer sequence (Pribnow box), seven in the spacer between the - 10 and -35 hexamer sequences, and the IS30 insertions just upstream of the -35 hexamer sequence. Four of the five substitution mutations changed a consensus base pair recognized by E sigma 70 RNA polymerase in the -10 or -35 hexamer. Decreased his expression caused by the 14 different his promoter mutations was measured in vivo. Relative to the wild-type promoter, the mutations resulted in as little as a 4-fold decrease to as much as a 357-fold decrease in his expression, with the largest decreases resulting from changes in the most highly conserved features of E sigma 70 promoters.     

The equilibrium constants and enthalpy changes for the esterification of glycine by ethanol in water and ethanol- water mixtures

Equilibrium constants, defined on the basis of the moles of all reactants and products including water, were found to be 4.6, 1.0 and 0.8 for the esterification of the glycinc cation at 20 degrees in a dilute aqueous sytem, and systems having 0.5 : 1 and 1 : 1 molar ratios of ethanol to water, respectively. When corrections were made for deviations from ideality a value of 5 +/- 1 was obtained for all three systems. Enthalpy changes were determined calorimetrically for the dilute aqueous and equimolar ethanol-water systems, and in each case was close to 1 kcal mole(-). The entropy change was calculated as 6 cal mole(-1) deg(-1).DeltaG(0') for the hydrolysis of the glycine ethyl ester cation at 20 degrees and pH 7 was calculated as -7.5 kcal mole(-1).     

Wildflower plantings promote blue orchard bee,Osmia lignaria (Hymenoptera: Megachilidae), reproduction in California almond orchards

Concerns over the availability of honeybees (Apis mellifera L.) to meet pollination demands have elicited interest in alternative pollinators to mitigate pressures on the commercial beekeeping industry. The blue orchard bee, Osmia lignaria (Say), is a commercially available native bee that can be employed as a copollinator with, or alternative pollinator to, honeybees in orchards. To date, their successful implementation in agriculture has been limited by poor recovery of bee progeny for use during the next spring. This lack of reproductive success may be tied to an inadequate diversity and abundance of alternative floral resources during the foraging period. Managed, supplementary wildflower plantings may promote O. lignaria reproduction in California almond orchards. Three wildflower plantings were installed and maintained along orchard edges to supplement bee forage. Plantings were seeded with native wildflower species that overlapped with and extended beyond almond bloom. We measured bee visitation to planted wildflowers, bee reproduction, and progeny outcomes across orchard blocks at variable distances from wildflower plantings during 2015 and 2016. Pollen provision composition was also determined to confirm O. lignaria wildflower pollen use. Osmia lignaria were frequently observed visiting wildflower plantings during, and after, almond bloom. Most O. lignaria nesting occurred at orchard edges. The greatest recovery of progeny occurred along the orchard edges having the closest proximity (80 m) to managed wildflower plantings versus edges farther away. After almond bloom, O. lignaria nesting closest to the wildflower plantings collected 72% of their pollen from Phacelia spp., which supplied 96% of the managed floral area. Phacelia spp. pollen collection declined with distance from the plantings, but still reached 17% 800 m into the orchard. This study highlights the importance of landscape context and proximity to supplementary floral resources in promoting the propagation of solitary bees as alternative managed pollinators in commercial agriculture.     

Effect of nematodes on rhizosphere colonization by seed-applied bacteria

There is much interest in the use of seed-applied bacteria for biocontrol and biofertilization, and several commercial products are available. However, many attempts to use this strategy fail because the seed-applied bacteria do not colonize the rhizosphere. Mechanisms of rhizosphere colonization may involve active bacterial movement or passive transport by percolating water or plant roots. Transport by other soil biota is likely to occur, but this area has not been well studied. We hypothesized that interactions with soil nematodes may enhance colonization. To test this hypothesis, a series of microcosm experiments was carried out using two contrasting soils maintained under well-defined physical conditions where transport by mass water flow could not occur. Seed-applied Pseudomonas fluorescens SBW25 was capable of rhizosphere colonization at matric potentials of -10 and -40 kPa in soil without nematodes, but colonization levels were substantially increased by the presence of nematodes. Our results suggest that nematodes can have an important role in rhizosphere colonization by bacteria in soil.     

Spatiotemporal variation in the reproductive ecology of two parapatric subspecies of Oenothera cespitosa (Onagraceae)

?Premise of the study: Flowering plants that rely on pollinators for most of their reproduction may experience unpredictable and inconsistent availability of effective pollinators throughout their reproductive lifetime. We investigated the reproductive ecology of two subspecies of the tufted evening primrose, Oenothera cespitosa, which occupy geographically and edaphically distinct habitats in western North America: O. cespitosa subsp. navajoensis inhabits sandstone soils on open sites or rocky slopes in the Colorado Plateau and O. cespitosa subsp. cespitosa grows in clay soils on talus slopes and exposed rocky ridges in the western Great Plains and northern Rocky Mountains of the United States. ?Methods: Pollen augmentation and selfing experiments, floral visitor observations, and single-visit effectiveness experiments were conducted over 4 years to examine the breeding system and spatiotemporal variation in pollinator behavior, assemblage, and abundance at different populations for each subspecies. ?Key results: Both subspecies of O. cespitosa were self-incompatible and pollen-limited, suggesting that the relative abundance, effectiveness, and movement patterns of different insects as pollinators influenced the quality and quantity of seed production in these plants. Medium-sized vespertine hawkmoths (Hyles lineata, Sphinx vashti) were effective pollinators when present, as were large matinal bees (Anthophora affabilis, A. dammersi, Xylocopa tabaniformis androleuca), whereas small oligolectic Lasioglossum bees primarily functioned as pollen thieves in the evening and morning. ?Conclusions: These findings highlight the importance of variability of pollinator composition and abundance in the evolution of plant breeding systems and reproductive success at varying spatial and temporal scales.     

Severe upper airway obstruction due to delayed retropharyngeal hematoma formation following blunt cervical trauma

Background

We report a case of severe upper airway obstruction due to a retropharyngeal hematoma that presented nearly one day after a precipitating traumatic injury. Retropharyngeal hematomas are rare, but may cause life-threatening airway compromise.

Case presentation

A 50 year-old man developed severe dyspnea with oropharyngeal airway compression due to retropharyngeal hematoma 20 hours after presenting to the emergency department. The patient also had a fractured first cervical vertebra and was diagnosed with a left brachial plexopathy. The patient underwent emergent awake fiberoptic endotracheal intubation to provide a definitive airway.

Conclusion

Retropharyngeal hematoma with life-threatening airway compromise can develop hours or days after a precipitating injury. Clinicians should be alert to the potential for this delayed airway collapse, and should also be prepared to rapidly secure the airway in this patient population likely to have concomitant cervical spinal or head injuries.     

Structural and Biochemical Basis for the Inhibitory Effect of Liprin-α3 on Mouse Diaphanous 1 (mDia1) Function

Diaphanous-related formins are eukaryotic actin nucleation factors regulated by an autoinhibitory interaction between the N-terminal RhoGTPase-binding domain (mDia_N) and the C-terminal Diaphanous-autoregulatory domain (DAD). Although the activation of formins by Rho proteins is well characterized, its inactivation is only marginally understood. Recently, liprin-α3 was shown to interact with mDia1. Overexpression of liprin-α3 resulted in a reduction of the cellular actin filament content. The molecular mechanisms of how liprin-α3 exerts this effect and counteracts mDia1 activation by RhoA are unknown. Here, we functionally and structurally define a minimal liprin-α3 core region, sufficient to recapitulate the liprin-α3 determined mDia1-respective cellular functions. We show that liprin-α3 alters the interaction kinetics and thermodynamics of mDia_N with RhoA·GTP and DAD. RhoA displaces liprin-α3 allosterically, whereas DAD competes with liprin-α3 for a highly overlapping binding site on mDia_N. Liprin-α3 regulates actin polymerization by lowering the regulatory potency of RhoA and DAD on mDia_N. We present a model of a mechanistically unexplored and new aspect of mDia_N regulation by liprin-α3.     

Mutations in the N‐terminal kinase‐like domain of the repressor of photomorphogenesis SPA1 severely impair SPA1 function but not light responsiveness in Arabidopsis

The COP1/SPA complex is an E3 ubiquitin ligase that acts as a key repressor of photomorphogenesis in dark‐grown plants. While both COP1 and the four SPA proteins contain coiled‐coil and WD‐repeat domains, SPA proteins differ from COP1 in carrying an N‐terminal kinase‐like domain that is not present in COP1. Here, we have analyzed the effects of deletions and missense mutations in the N‐terminus of SPA1 when expressed in a spa quadruple mutant background devoid of any other SPA proteins. Deletion of the large N‐terminus of SPA1 severely impaired SPA1 activity in transgenic plants with respect to seedling etiolation, leaf expansion and flowering time. This ΔN SPA1 protein showed a strongly reduced affinity for COP1 in vitro and in vivo, indicating that the N‐terminus contributes to COP1/SPA complex formation. Deletion of only the highly conserved 95 amino acids of the kinase‐like domain did not severely affect SPA1 function nor interactions with COP1 or cryptochromes. In contrast, missense mutations in this part of the kinase‐like domain severely abrogated SPA1 function, suggesting an overriding negative effect of these mutations on SPA1 activity. We therefore hypothesize that the sequence of the kinase‐like domain has been conserved during evolution because it carries structural information important for the activity of SPA1 in darkness. The N‐terminus of SPA1 was not essential for light responsiveness of seedlings, suggesting that photoreceptors can inhibit the COP1/SPA complex in the absence of the SPA1 N‐terminal domain. Together, these results uncover an important, but complex role of the SPA1 N‐terminus in the suppression of photomorphogenesis.     

Long term repeated prescribed burning increases evenness in the basidiomycete laccase gene pool in forest soils

Repeated prescribed burning alters the biologically labile fraction of nutrients and carbon of soil organic matter (SOM). Using a long-term (30 years) repeated burning experiment where burning has been carried out at a 2- or 4-year frequency, we analysed the effect of prescribed burning on gross potential C turnover rates and phenol oxidase activity in relation to shifts in SOM composition as observed using Fourier-transform infrared spectroscopy. In tandem, we assessed the genetic diversity of basidiomycete laccases. While the overall effect of burning was a decline in phenol oxidase activity, Shannon diversity and evenness of laccases was significantly higher in burned sites. Co-correspondence analysis of SOM composition and laccase operational taxonomic unit frequency data also suggested a strong correlation. While this correlation could indicate that the observed increase in laccase genetic diversity due to burning is due to increased resource diversity, a temporal replacement of the most abundant members of the assembly by an otherwise dormant pool of fungi cannot be excluded. As such, our results fit the intermediate disturbance hypothesis. Effects were stronger in plots burned in 2-year rotations, suggesting that the 4-year burn frequency may be a more sustainable practice to ensure the long-term stability of C cycling in such ecosystems.