Histone H2A and H4 N-terminal Tails Are Positioned by the MEP50 WD Repeat Protein for Efficient Methylation by the PRMT5 Arginine Methyltransferase

The protein arginine methyltransferase PRMT5 is complexed with the WD repeat protein MEP50 (also known as Wdr77 or androgen coactivator p44) in vertebrates in a tetramer of heterodimers. MEP50 is hypothesized to be required for protein substrate recruitment to the catalytic domain of PRMT5. Here we demonstrate that the cross-dimer MEP50 is paired with its cognate PRMT5 molecule to promote histone methylation. We employed qualitative methylation assays and a novel ultrasensitive continuous assay to measure enzyme kinetics. We demonstrate that neither full-length human PRMT5 nor the Xenopus laevis PRMT5 catalytic domain has appreciable protein methyltransferase activity. We show that histones H4 and H3 bind PRMT5-MEP50 more efficiently compared with histone H2A(1–20) and H4(1–20) peptides. Histone binding is mediated through histone fold interactions as determined by competition experiments and by high density histone peptide array interaction studies. Nucleosomes are not a substrate for PRMT5-MEP50, consistent with the primary mode of interaction via the histone fold of H3-H4, obscured by DNA in the nucleosome. Mutation of a conserved arginine (Arg-42) on the MEP50 insertion loop impaired the PRMT5-MEP50 enzymatic efficiency by increasing its histone substrate K_m, comparable with that of Caenorhabditis elegans PRMT5. We show that PRMT5-MEP50 prefers unmethylated substrates, consistent with a distributive model for dimethylation and suggesting discrete biological roles for mono- and dimethylarginine-modified proteins. We propose a model in which MEP50 and PRMT5 simultaneously engage the protein substrate, orienting its targeted arginine to the catalytic site.     

Life-cycle modification in open oceans accounts for genome variability in a cosmopolitan phytoplankton

Emiliania huxleyi is the most abundant calcifying plankton in modern oceans with substantial intraspecific genome variability and a biphasic life cycle involving sexual alternation between calcified 2N and flagellated 1N cells. We show that high genome content variability in Emiliania relates to erosion of 1N-specific genes and loss of the ability to form flagellated cells. Analysis of 185 E. huxleyi strains isolated from world oceans suggests that loss of flagella occurred independently in lineages inhabiting oligotrophic open oceans over short evolutionary timescales. This environmentally linked physiogenomic change suggests life cycling is not advantageous in very large/diluted populations experiencing low biotic pressure and low ecological variability. Gene loss did not appear to reflect pressure for genome streamlining in oligotrophic oceans as previously observed in picoplankton. Life-cycle modifications might be common in plankton and cause major functional variability to be hidden from traditional taxonomic or molecular markers.     

Primary Succession on a Hawaiian Dryland Chronosequence

We used measurements from airborne imaging spectroscopy and LiDAR to quantify the biophysical structure and composition of vegetation on a dryland substrate age gradient in Hawaii. Both vertical stature and species composition changed during primary succession, and reveal a progressive increase in vertical stature on younger substrates followed by a collapse on Pleistocene-aged flows. Tall-stature Metrosideros polymorpha woodlands dominated on the youngest substrates (hundreds of years), and were replaced by the tall-stature endemic tree species Myoporum sandwicense and Sophora chrysophylla on intermediate-aged flows (thousands of years). The oldest substrates (tens of thousands of years) were dominated by the short-stature native shrub Dodonaea viscosa and endemic grass Eragrostis atropioides. We excavated 18 macroscopic charcoal fragments from Pleistocene-aged substrates. Mean radiocarbon age was 2,002 years and ranged from < 200 to 7,730. Genus identities from four fragments indicate that Osteomeles spp. or M. polymorpha once occupied the Pleistocene-aged substrates, but neither of these species is found there today. These findings indicate the existence of fires before humans are known to have occupied the Hawaiian archipelago, and demonstrate that a collapse in vertical stature is prevalent on the oldest substrates. This work contributes to our understanding of prehistoric fires in shaping the trajectory of primary succession in Hawaiian drylands.     

Factors Influencing the Survival of Sympatric Gorilla (<Emphasis Type="Italic">Gorilla gorilla gorilla</Emphasis>) and Chimpanzee (<Emphasis Type="Italic">Pan troglodytes troglodytes</Emphasis>) Nests

Accurate and precise surveys of primate abundance provide the basis for understanding species ecology and essential information for conservation assessments. Owing to the elusive nature of wild apes and the vast region of dense forest they inhabit, population estimates of central chimpanzees (Pan troglodytes troglodytes) and western lowland gorillas (Gorilla gorilla gorilla) have largely relied on surveys of their nests. Specific information about the nesting behavior of apes permits the estimation of the number of nests built (nest creation rate). Similarly, information on nest characteristics and environmental factors can be used to estimate the time it takes nests to decay (nest decay rate). Nest creation and decay rates are then used to convert nest density estimates to absolute ape densities. Population estimates that use site-specific estimates of nest creation and decay rates are more accurate and precise. However, it is common practice to generalize these conversion factors across sites because of the additional cost of studies required to gather the information to estimate them. Over a 9-mo study period, we detected and monitored the time to decay of gorilla nests (N = 514) and chimpanzee nests (N = 521) in northern Republic of Congo. We investigated the influence of nest characteristics and environmental factors on nest survivorship and estimated the mean time to nest decay (or equivalently survival) using MARK. Key factors influencing nest decay rate included ape species, forest type, nest height, mean rainfall, nest structure, nest type, and primary aspects of nest construction. Our findings highlight the synergistic effect of behavior and environment on great ape nest degradation, as well as providing practical insights for improving measures to monitor remaining populations of these endangered species.     

Evaluation of liquid and solid culture media for the recovery and enrichment of Burkholderia cenocepacia from distilled water

Burkholderia cepacia complex (BCC) presence has been the cause of recalls of both sterile and non-sterile pharmaceutical products since these opportunistic pathogens have been implicated to cause infections to susceptible individuals. BCC are ubiquitous in nature, but in pharmaceutical settings the most common source is contaminated water systems. Some strains of BCC, previously described as Pseudomonas cepacia, were not readily detected by standard culture methods. We have explored different strategies to recover and enrich Burkholderia cenocepacia previously cultured in distilled water for 40 days. Enrichment media of varied nutrient concentrations and composition were used, including modified Tryptic Soy Agar or Broth (TSA or TSB), Reasoner’s 2nd Agar or Broth (R2A or R2AB), Brain–Heart Infusion Broth (BHIB), Mueller–Hinton Broth (MHB), and Ashdown’s (ASH) medium. Of the various broth media tested, cell growth was significantly greater in TSB and R2AB than in BHIB, MHB, or ASH broth. TSB and R2AB were also compared for their recovery efficiency. Generally, there was no significant difference between the numbers of B. cenocepacia grown on 15 differently modified TSA and five modified R2A solid media. Overall, however, diluted TSA and TSB media, and R2A and R2AB showed better recovery efficiency than TSA and TSB for inocula containing small numbers of cells. All strains persisted in distilled water for 40 days. Broth media were more effective than solid media for recovery of B. cenocepacia from distilled water. These results may assist in improving detection assays with recovery and enrichment strategies to maximize recovery of these fastidious organisms.     

Characterization of the cydAB-Encoded Cytochrome bd Oxidase from Mycobacterium smegmatis

The cydAB genes from Mycobacterium smegmatis have been cloned and characterized. The cydA and cydB genes encode the two subunits of a cytochrome bd oxidase belonging to the widely distributed family of quinol oxidases found in prokaryotes. The cydD and cydC genes located immediately downstream of cydB encode a putative ATP-binding cassette-type transporter. At room temperature, reduced minus oxidized difference spectra of membranes purified from wild-type M. smegmatis displayed spectral features that are characteristic of the gamma-proteobacterial type cytochrome bd oxidase. Inactivation of cydA or cydB by insertion of a kanamycin resistance marker resulted in loss of d-heme absorbance at 631 nm. The d-heme could be restored by transformation of the M. smegmatis cyd mutants with a replicating plasmid carrying the highly homologous cydABDC gene cluster from Mycobacterium tuberculosis. Inactivation of cydA had no effect on the ability of M. smegmatis to exit from stationary phase at 37 or 42 degrees C. The growth rate of the cydA mutant was tested under oxystatic conditions. Although no discernible growth defect was observed under moderately aerobic conditions (9.2 to 37.5 x 10(2) Pa of pO(2) or 5 to 21% air saturation), the mutant displayed a significant growth disadvantage when cocultured with the wild type under extreme microaerophilia (0.8 to 1.7 x 10(2) Pa of pO(2) or 0.5 to 1% air saturation). These observations were in accordance with the two- to threefold increase in cydAB gene expression observed upon reduction of the pO(2) of the growth medium from 21 to 0.5% air saturation and with the concomitant increase in d-heme absorbance in spectra of membranes isolated from wild-type M. smegmatis cultured at 1% air saturation. Finally, the cydA mutant displayed a competitive growth disadvantage in the presence of the terminal oxidase inhibitor, cyanide, when cocultured with wild type at 21% air saturation in an oxystat. In conjunction with these findings, our results suggest that cytochrome bd is an important terminal oxidase in M. smegmatis.     

Induction time for resistance to microsporidial gill disease caused by Loma salmonae following vaccination of rainbow trout (Oncorhynchus mykiss) with a spore-based vaccine

Resistance to re-infection of rainbow trout to Loma salmonae, a microsporidian gill parasite has been previously documented and this study examined how rapidly this resistance develops. Naive rainbow trout were inoculated intraperitoneally (IP) with an inactivated spore-based vaccine and were then given an oral challenge with a high dose of L. salmonae spores at various weeks after being vaccinated. Non-vaccinated naive fish (exposed group) were challenged alongside of each group of vaccinated fish to ensure that the challenges were relatively standardised. In each group of fish, four weeks after the challenge, numbers of xenomas were counted on a gill arch for all fish. Vaccinated trout were completely resistant to a L. salmonae challenge six weeks after vaccination, although the onset of resistance began at approximately week 3, as observed with a reduction in the percent infected and xenoma intensity. The maximum percent infected for the vaccinated fish was 83% following a challenge two weeks following vaccination, whereas for the exposed group the maximum prevalence of 100% was reached several times. With continued research, a spore-based vaccine for L. salmonae has the potential to become the first commercially available parasite vaccine for fish.     

Actin redistribution in mosquito malpighian tubules after a blood meal and cyclic AMP stimulation

Fluid secretion by mosquito Malpighian tubules is critical to maintaining fluid and electrolyte balance after a blood meal. Endogenous cAMP levels increase in Malpighian tubules after a blood meal. Here, we determined if corresponding changes in intracellular actin distribution occur after a blood meal or dibutyryl-cAMP (db-cAMP) stimulation and whether altering actin turnover inhibits secretion. In untreated Malpighian tubules, beta-actin immunostaining was more intense in the apical region of adult Malpighian tubules than in the cytoplasm. Stimulation by a blood meal or db-cAMP significantly decreased beta-actin immunostaining in the non-apical region of the cell. Db-cAMP had similar effects in larvae and pupae Malpighian tubules. In contrast, no detectable shift in F-actin distribution was detected; however, F-actin bundles within the cytoplasm increased in size after treatment with db-cAMP. Pretreatment of Malpighian tubules with agents perturbing actin fiber assembly and disassembly decreased basal secretion rates and inhibited the stimulatory effects of db-cAMP. Our results show (1) beta-actin redistributes toward the apical membrane after a blood meal and this correlates temporally with increase urine flow rate and intracellular cAMP levels, (2) Malpighian tubules from all developmental stages exhibit this same response to db-cAMP-stimulation, and (3) dynamic assembly and disassembly of beta-actin is required for db-cAMP-stimulated secretion.