Proteus mirabilis interkingdom swarming signals attract blow flies

Flies transport specific bacteria with their larvae that provide a wider range of nutrients for those bacteria. Our hypothesis was that this symbiotic interaction may depend on interkingdom signaling. We obtained Proteus mirabilis from the salivary glands of the blow fly Lucilia sericata; this strain swarmed significantly and produced a strong odor that attracts blow flies. To identify the putative interkingdom signals for the bacterium and flies, we reasoned that as swarming is used by this bacterium to cover the food resource and requires bacterial signaling, the same bacterial signals used for swarming may be used to communicate with blow flies. Using transposon mutagenesis, we identified six novel genes for swarming (ureR, fis, hybG, zapB, fadE and PROSTU_03490), then, confirming our hypothesis, we discovered that fly attractants, lactic acid, phenol, NaOH, KOH and ammonia, restore swarming for cells with the swarming mutations. Hence, compounds produced by the bacterium that attract flies also are utilized for swarming. In addition, bacteria with the swarming mutation rfaL attracted fewer blow flies and reduced the number of eggs laid by the flies. Therefore, we have identified several interkingdom signals between P. mirabilis and blow flies.     

A comparison of assisted and unassisted proprioceptive neuromuscular facilitation techniques and static stretching

A comparison of assisted and unassisted proprioceptive neuromuscular facilitation techniques and static stretching. J Strength Cond Res 26(5): 1238-1244, 2012-Proprioceptive neuromuscular facilitation (PNF) stretching often requires a partner. Straps are available allowing an individual to perform PNF stretching alone. It is not known if a strap provides similar improvements in the range of motion (ROM) as partner-assisted PNF or static stretching. The purpose of this study was to compare assisted and unassisted (with a strap) PNF stretching and static stretching. Hip joint ROM, reaction time (RT), and movement time (MT) were measured prestretching and poststretching. Thirteen recreationally active adults participated in this study. The participants were subjected to 5 different stretch interventions in a random order on separate days. Stretch conditions included unassisted PNF stretching using (a) isometric, (b) concentric, and (c) eccentric contractions with a stretch strap, (d) partner-assisted isometric PNF, and (e) static stretching. The RT, MT, dynamic, active, passive hip flexion angle, and angular velocity with dynamic hip flexion were measured before and after the intervention. The ROM improved (p < 0.05) 2.6, 2.7, and 5.4%, respectively, with dynamic, active static, and passive static ROM, but there was no significant difference between the stretching protocols. There was a main effect for time (p < 0.05) with all stretching conditions negatively impacting dynamic angular velocity (9.2%). Although there was no significant effect on RT, MT showed a negative main effect for time (p < 0.05) slowing 3.4%. In conclusion, it was found that all 3 forms of active stretching provided similar improvements in the ROM and poststretching performance decrements in MT and angular velocity. Thus, individuals can implement PNF stretching techniques with a partner or alone with a strap to improve ROM, but athletes should not use these techniques before important competitions or training because of the impairment of limb velocity and MT.     

Identification of super-infected Aedes triseriatus mosquitoes collected as eggs from the field and partial characterization of the infecting La Crosse viruses

Background

La Crosse virus (LACV) is a pathogenic arbovirus that is transovarially transmitted by Aedes triseriatus mosquitoes and overwinters in diapausing eggs. However, previous models predicted transovarial transmission (TOT) to be insufficient to maintain LACV in nature.

Results

To investigate this issue, we reared mosquitoes from field-collected eggs and assayed adults individually for LACV antigen, viral RNA by RT-PCR, and infectious virus. The mosquitoes had three distinct infection phenotypes: 1) super infected (SI+) mosquitoes contained infectious virus, large accumulations of viral antigen and RNA and comprised 17 of 17,825 (0.09%) of assayed mosquitoes, 2) infected mosquitoes (I+) contained no detectable infectious virus, lesser amounts of viral antigen and RNA, and comprised 3.7% of mosquitoes, and 3) non-infected mosquitoes (I-) contained no detectable viral antigen, RNA, or infectious virus and comprised 96.21% of mosquitoes. SI+ mosquitoes were recovered in consecutive years at one field site, suggesting that lineages of TOT stably-infected and geographically isolated Ae. triseriatus exist in nature. Analyses of LACV genomes showed that SI+ isolates are not monophyletic nor phylogenetically distinct and that synonymous substitution rates exceed replacement rates in all genes and isolates. Analysis of singleton versus shared mutations (Fu and Li's F*) revealed that the SI+ LACV M segment, with a large and significant excess of intermediate-frequency alleles, evolves through disruptive selection that maintains SI+ alleles at higher frequencies than the average mutation rate. A QTN in the LACV NSm gene was detected in SI+ mosquitoes, but not in I+ mosquitoes. Four amino acid changes were detected in the LACV NSm gene from SI+ but not I+ mosquitoes from one site, and may condition vector super infection. In contrast to NSm, the NSs sequences of LACV from SI+ and I+ mosquitoes were identical.

Conclusions

SI+ mosquitoes may represent stabilized infections of Ae. triseriatus mosquitoes, which could maintain LACV in nature. A gene-for-gene interaction involving the viral NSm gene and a vector innate immune response gene may condition stabilized infection.     

Screening microbiota for effects on host tissues

The assembly and function of microbial communities depends on many factors including the local environment and the metabolic properties of the colonizing organisms. Chemical communications or other secreted factors also play a role and are used by different microbial strains both cooperatively and competitively. The spectrum of microbial secretions have various effects on the microbe's respective hosts, both positive and negative. Thus, characterizing the roles of microbial community members and their secretions can yield key mechanistic insights into microbiome function and can lead to new intervention strategies. Focusing on the simple, yet important functional impact of toxicity, we quantify supernatant dosage responses with image data and examine the morphological effects of microbial secretions on skin-associated host cells. Since the diversity of microbial communities, coupled with the multiplicity of host tissues requires scalable methods, we develop and demonstrate a microfluidic device that enables high-content screening of microbial secretion effects on adherent cell types.     

Differential regulation of mouse equilibrative nucleoside transporter 1 (mENT1) splice variants by protein kinase CK2

Nucleosides are accumulated by cells via a family of equilibrative transport proteins (ENTs). An alternative splice variant of the most common subtype of mouse ENT (ENT1) has been identified which is missing a protein kinase CK2 (casein kinase 2) consensus site (Ser²⁵⁴) in the central intracellular loop of the protein. We hypothesized that this variant (mENT1a) would be less susceptible to modulation by CK2-mediated phosphorylation compared to the variant containing the serine at position 254 (mENT1b). Each splice variant was transfected into nucleoside transporter deficient PK15 cells, and stable transfectants assessed for their ability to bind the ENT1-selective probe [³H]nitrobenzylthioinosine (NBMPR) and to mediate the cellular uptake of [³H]2-chloroadenosine, with or without treatment with the CK2 selective inhibitor, 4,5,6,7-tetrabromobenzotriazole (TBB). mENT1a had a higher affinity for NBMPR relative to mENT1b – measured both directly by the binding of [³H]NBMPR, and indirectly via inhibition of [³H]2-chloroadenosine influx by NBMPR. Furthermore, incubation of mENT1b-expressing cells with 10 µM TBB for 48 h decreased both the K_D and B_max of [³H]NBMPR binding, as well as the V_max of 2-chloroadenosine uptake, whereas similar treatment of mENT1a-expressing cells with TBB had no effect. PK15 cells transfected with hENT1, which has Ser²⁵⁴, was similar to mENT1b in its response to TBB. In conclusion, inhibition of CK2 activity, or deletion of Ser²⁵⁴ from mENT1, enhances transporter affinity for the inhibitor, NBMPR, and reduces the number of ENT1 proteins functioning at the level of the plasma membrane.     

Reduced Heme Levels Underlie the Exponential Growth Defect of the Shewanella oneidensis hfq Mutant

The RNA chaperone Hfq fulfills important roles in small regulatory RNA (sRNA) function in many bacteria. Loss of Hfq in the dissimilatory metal reducing bacterium Shewanella oneidensis strain MR-1 results in slow exponential phase growth and a reduced terminal cell density at stationary phase. We have found that the exponential phase growth defect of the hfq mutant in LB is the result of reduced heme levels. Both heme levels and exponential phase growth of the hfq mutant can be completely restored by supplementing LB medium with 5-aminolevulinic acid (5-ALA), the first committed intermediate synthesized during heme synthesis. Increasing expression of gtrA, which encodes the enzyme that catalyzes the first step in heme biosynthesis, also restores heme levels and exponential phase growth of the hfq mutant. Taken together, our data indicate that reduced heme levels are responsible for the exponential growth defect of the S. oneidensis hfq mutant in LB medium and suggest that the S. oneidensis hfq mutant is deficient in heme production at the 5-ALA synthesis step.     

N2-fixation by methanotrophs sustains carbon and nitrogen accumulation in pristine peatlands

Symbiotic relationships between N₂-fixing prokaryotes and their autotrophic hosts are essential in nitrogen (N)-limited ecosystems, yet the importance of this association in pristine boreal peatlands, which store 25 % of the world’s soil (C), has been overlooked. External inputs of N to bogs are predominantly atmospheric, and given that regions of boreal Canada anchor some of the lowest rates found globally (~1 kg N ha^?1 year^?1), biomass production is thought to be limited primarily by N. Despite historically low N deposition, we show that boreal bogs have accumulated approximately 12–25 times more N than can be explained by atmospheric inputs. Here we demonstrate high rates of biological N₂-fixation in prokaryotes associated with Sphagnum mosses that can fully account for the missing input of N needed to sustain high rates of C sequestration. Additionally, N amendment experiments in the field did not increase Sphagnum production, indicating that mosses are not limited by N. Lastly, by examining the composition and abundance of N₂-fixing prokaryotes by quantifying gene expression of 16S rRNA and nitrogenase-encoding nifH, we show that rates of N₂-fixation are driven by the substantial contribution from methanotrophs, and not from cyanobacteria. We conclude biological N₂-fixation drives high sequestration of C in pristine peatlands, and may play an important role in moderating fluxes of methane, one of the most important greenhouse gases produced in peatlands. Understanding the mechanistic controls on biological N₂-fixation is crucial for assessing the fate of peatland carbon stocks under scenarios of climate change and enhanced anthropogenic N deposition.     

High-resolution paramagnetically enhanced solid-state NMR spectroscopy of membrane proteins at fast magic angle spinning

Magic angle spinning nuclear magnetic resonance (MAS NMR) is well suited for the study of membrane proteins in membrane mimetic and native membrane environments. These experiments often suffer from low sensitivity, due in part to the long recycle delays required for magnetization and probe recovery, as well as detection of low gamma nuclei. In ultrafast MAS experiments sensitivity can be enhanced through the use of low power sequences combined with paramagnetically enhanced relaxation times to reduce recycle delays, as well as proton detected experiments. In this work we investigate the sensitivity of ¹³C and ¹H detected experiments applied to 27 kDa membrane proteins reconstituted in lipids and packed in small 1.3 mm MAS NMR rotors. We demonstrate that spin diffusion is sufficient to uniformly distribute paramagnetic relaxation enhancement provided by either covalently bound or dissolved CuEDTA over 7TM alpha helical membrane proteins. Using paramagnetic enhancement and low power decoupling in carbon detected experiments we can recycle experiments ~13 times faster than under traditional conditions. However, due to the small sample volume the overall sensitivity per unit time is still lower than that seen in the 3.2 mm probe. Proton detected experiments, however, showed increased efficiency and it was found that the 1.3 mm probe could achieve sensitivity comparable to that of the 3.2 mm in a given amount of time. This is an attractive prospect for samples of limited quantity, as this allows for a reduction in the amount of protein that needs to be produced without the necessity for increased experimental time.