Lipid metabolism of phenol-tolerant <Emphasis Type="Italic">Rhodococcus opacus</Emphasis> strains for lignin bioconversion

Background

Lignin is a recalcitrant aromatic polymer that is a potential feedstock for renewable fuel and chemical production. Rhodococcus opacus PD630 is a promising strain for the biological upgrading of lignin due to its ability to tolerate and utilize lignin-derived aromatic compounds. To enhance its aromatic tolerance, we recently applied adaptive evolution using phenol as a sole carbon source and characterized a phenol-adapted R. opacus strain (evol40) and the wild-type (WT) strain by whole genome and RNA sequencing. While this effort increased our understanding of the aromatic tolerance, the tolerance mechanisms were not completely elucidated.

Results

We hypothesize that the composition of lipids plays an important role in phenol tolerance. To test this hypothesis, we applied high-resolution mass spectrometry analysis to lipid samples obtained from the WT and evol40 strains grown in 1 g/L glucose (glucose), 0.75 g/L phenol (low phenol), or 1.5 g/L phenol (high phenol, evol40 only) as a sole carbon source. This analysis identified?>?100 lipid species of mycolic acids, phosphatidylethanolamines (PEs), phosphatidylinositols (PIs), and triacylglycerols. In both strains, mycolic acids had fewer double bond numbers in phenol conditions than the glucose condition, and evol40 had significantly shorter mycolic acid chain lengths than the WT strain in phenol conditions. These results indicate that phenol adaptation affected mycolic acid membrane composition. In addition, the percentage of unsaturated phospholipids decreased for both strains in phenol conditions compared to the glucose condition. Moreover, the PI content increased for both strains in the low phenol condition compared to the glucose condition, and the PI content increased further for evol40 in the high phenol condition relative to the low phenol condition.

Conclusions

This work represents the first comprehensive lipidomic study on the membrane of R. opacus grown using phenol as a sole carbon source. Our results suggest that the alteration of the mycolic acid and phospholipid membrane composition may be a strategy of R. opacus for phenol tolerance.

     

Subtle variation in size and shape of the whole forewing and the red band among co‐mimics revealed by geometric morphometric analysis in Heliconius butterflies

Heliconius are unpalatable butterflies that exhibit remarkable intra‐ and interspecific variation in wing color pattern, specifically warning coloration. Species that have converged on the same pattern are often clustered in Müllerian mimicry rings. Overall, wing color patterns are nearly identical among co‐mimics. However, fine‐scale differences exist, indicating that factors in addition to natural selection may underlie wing phenotype. Here, we investigate differences in shape and size of the forewing and the red band in the Heliconius postman mimicry ring (H. erato phyllis and the co‐mimics H. besckei, H. melpomene burchelli, and H. melpomene nanna) using a landmark‐based approach. If phenotypic evolution is driven entirely by predation pressure, we expect nonsignificant differences among co‐mimics in terms of wing shape. Also, a reinforcement of wing pattern (i.e., greater similarity) could occur when co‐mimics are in sympatry. We also examined variation in the red forewing band because this trait is critical for both mimicry and sexual communication. Morphometric results revealed significant but small differences among species, particularly in the shape of the forewing of co‐mimics. Although we did not observe greater similarity when co‐mimics were in sympatry, nearly identical patterns provided evidence of convergence for mimicry. In contrast, mimetic pairs could be distinguished based on the shape (but not the size) of the red band, suggesting an “advergence” process. In addition, sexual dimorphism in the red band shape (but not size) was found for all lineages. Thus, we infer that natural selection due to predation by birds might not be the only mechanism responsible for variation in color patterns, and sexual selection could be an important driver of wing phenotypic evolution in this mimicry ring.     

Testing the AC/DC hypothesis: Rock and roll is noise pollution and weakens a trophic cascade

Anthropogenic sound is increasingly considered a major environmental issue, but its effects are relatively unstudied. Organisms may be directly affected by anthropogenic sound in many ways, including interference with their ability to detect mates, predators, or food, and disturbances that directly affect one organism may in turn have indirect effects on others. Thus, to fully appreciate the net effect of anthropogenic sound, it may be important to consider both direct and indirect effects. We report here on a series of experiments to test the hypothesis that anthropogenic sound can generate cascading indirect effects within a community. We used a study system of lady beetles, soybean aphids, and soybean plants, which are a useful model for studying the direct and indirect effects of global change on food webs. For sound treatments, we used several types of music, as well as a mix of urban sounds (e.g., sirens, vehicles, and construction equipment), each at volumes comparable to a busy city street or farm tractor. In 18‐hr feeding trials, rock music and urban sounds caused lady beetles to consume fewer aphids, but other types of music had no effect even at the same volume. We then tested the effect of rock music on the strength of trophic cascades in a 2‐week experiment in plant growth chambers. When exposed to music by AC/DC, who articulated the null hypothesis that “rock and roll ain't noise pollution” in a song of the same name, lady beetles were less effective predators, resulting in higher aphid density and reduced final plant biomass relative to control (no music) treatments. While it is unclear what characteristics of sound generate these effects, our results reject the AC/DC hypothesis and demonstrate that altered interspecific interactions can transmit the indirect effects of anthropogenic noise through a community.     

p-Chlorophenylalanine Changes Serotonin Transporter mRNA Levels and Expression of the Gene Product

Abstract: After a single intraperitoneal injection of the irreversible tryptophan hydroxylase inhibitor p -chlorophenylalanine (PCPA; 300 mg/kg), there was a rapid down-regulation of serotonin (5-HT) transporter mRNA levels in cell bodies. This change was significant at 1 and 2 days after PCPA administration within the ventromedial but not the dorsomedial portion of the dorsal raphe nucleus. Seven days after PCPA treatment, 5-HT transporter mRNA levels were significantly elevated compared with controls in both regions of the dorsal raphe nucleus. PCPA administration produced no change in the [³H]-citalopram binding and synaptosomal [³H]5-HT uptake in terminal regions at 2 and 7 days after treatment but significantly reduced both these parameters by ∼20% in the hippocampus and in cerebral cortex 14 days after PCPA administration. The striatum showed a lower sensitivity to this effect. No significant changes were observed in the levels of [³H]citalopram binding to 5-HT cell bodies in the dorsal raphe nucleus. In the same animals used for 5-HT transporter mRNA level measurements, levels of tryptophan hydroxylase mRNA in neurons of the ventromedial and dorsomedial portions of the dorsal raphe nucleus were increased 2 days after PCPA administration and fell to control levels 7 days after injection in the ventromedial region but not in the dorsomedial portion of the dorsal raphe nucleus, where they remained significantly higher than controls. Altogether, these results show that changes in 5-HT transporter mRNA are not temporally related to changes in 5-HT transporter protein levels. In addition, our results suggest that the 5-HT transporter and tryptophan hydroxylase genes are regulated by different mechanisms. We also provide further evidence that dorsal raphe 5-HT neurons are differentially regulated by drugs, depending on their location.     

DIDS increases K+ secretion through an I sKchannel in apical membrane of vestibular dark cell epithelium of gerbil

Vestibular dark cell epithelium secretes K⁺ via I _sKchannels in the apical membrane. The previous observation that disulfonic stilbenes increased the equivalent short circuit current (I _sc) suggested that these agents might be useful investigative tools in this tissue. The present experiments were conducted to determine if the increase in I _scwas associated with an increase in K⁺ flux and if the effect was directly on the I _sKchannel or indirectly via a cytosolic intermediary. Measurements of transepithelial K⁺ flux with the K⁺-selective vibrating probe and of changes in net cellular solute flux by measurements of epithelial cell height showed that 4,4-diisothiocyanatostilbene-2,2-disulfonic acid (DIDS) increased K⁺ flux by a factor of 1.96±0.71 and caused net solute efflux. The apical membrane was partitioned with a macropatch pipette and DIDS was applied either to the membrane outside the pipette, inside the pipette or to the entire apical membrane. DIDS inside the pipette increased the current across the patch, the membrane conductance, the slowly-inactivating (I _sK) component of the membrane current and shifted the reversal voltage toward the equilibrium potential for K⁺. DIDS outside the patch decreased the patch current and conductance, consistent with shunting of current away from the membrane patch. These findings strongly support the notion that DIDS increases K⁺ secretion through I _sKchannels in the apical membrane of vestibular dark cell epithelium by acting directly on the channels or on a tightly colocalized membrane component.We thank Dr. Peter J.S. Smith and Alan Shipley of the National Vibrating Probe Facility at the Marine Biological Laboratory at Woods Hole, MA for their support and assistance in the measurements of K⁺ flux. This work was supported by National Institutes of Health grants R01-DC00212, R29-DC1098 and P41-RR01395.     

Notes on the geographic distribution of howling monkeys in the marajó archipelago, pará, Brazil

I review the distribution of genus Alouattain the Marajó Archipelago based on geographic variation of pelage color patterns. Specific differences exist among the labeled specimens at Museu Paraense Emílio Goeldi, which are confirmed via new field information from wild populations. The animals from Marajó, Caviana, and Mexiana Islands possess color patterns of Alouatta belzebul,while Alouatta seniculusis confirmed as the species that inhabits Gurupá Island. The high variability of pelage coloration of the animals from Marajó, Caviana, and Mexiana Islands includes all phenotypes formerly designated as different subspecies. The howler populations from these three islands belong to the same subspecies as those populations that inhabit the region of Tucurui dam reservoir (area 4). Finally, it seems that, as also suggested by chromosome studies, A. belzebulprobably comprises different epiphenotypes without taxonomic validity.     

The effects of pond duration on the life history traits of an ephemeral pond crustacean,Eulimnadia texana

We examined the relationship between pond duration and life history characters of the clam shrimp Eulimnadia texana, a species inhabiting ephemeral ponds in southwestern North America. Since the shrimp live in temporary habitats, we predicted that there should be high selection pressure on life history characteristics associated with rapid development (e.g., fast growth, early maturity, etc.), rather than selection for increased longevity. Pond duration was estimated using a combination of average monthly rainfall and pond size (surface to volume ratio). Shrimp that live in smaller ponds (high surface to volume ratio)in areas with low average rainfall should, on average, experience a shorter total time available for development than those in larger ponds or in areas of higher rainfall. These shrimp should have an earlier age at maturity, reduced longevity, lower fecundity, and faster growth. Five replicate populations of clam shrimp were collected as cysts from five ponds. These shrimp were raised in a common garden experiment in the laboratory. Daily measurements of growth and egg production were taken and ages at maturity and death were recorded. Shrimp from areas with higher average rainfall had slower growth, higher fecundity, greater longevity, and an earlier age at maturity than those from areas with lower average rainfall. If average rainfall is an accurate measure of pond duration, then the first three of these life history traits differ in the directions expected. However, age at maturity varied in a manner opposite to that expected, being earlier in the ponds with longer duration. Surface to volume ratio was not helpful in further resolving differences in these life history characters. This revised version was published online in August 2006 with corrections to the Cover Date.     

Inhibition of the High-Affinity Brain Glutamate Transporter GLAST-1 via Direct Phosphorylation

Abstract: Neurotransmission at excitatory glutamatergic synapses is terminated by the reuptake of the neurotransmitter by high-affinity transporters, which keep the extracellular glutamate concentration below excitotoxic levels. The amino acid sequence of the recently isolated and cloned brain-specific glutamate/aspartate transporter (GLAST-1) of the rat reveals three consensus sequences of putative phosphorylation sites for protein kinase C (PKC). The PKC activator phorbol 12-myristate 13-acetate (PMA) decreased glutamate transport activity in Xenopus oocytes and human embryonic kidney cells (HEK293) expressing the cloned GLAST-1 cDNA, within 20 min, to 25% of the initial transport activity. This down-regulation was blocked by the PKC inhibitor staurosporine. GLAST-1 transport activity remains unimpaired by phorbol 12-monomyristate. Removal of all putative PKC sites of wild-type GLAST-1 by site-directed mutagenesis did not abolish inhibition of glutamate transport. [³²P]Phosphate-labeled wild-type and mutant transport proteins devoid of all predicted PKC sites were detected by immunoprecipitation after stimulation with PMA. Immunoprecipitation of [³⁵S]methionine-labeled transporter molecules indicates a similar stability of phosphorylated and nonphosphorylated GLAST-1 protein. Immunofluorescence staining did not differentiate surface staining of HEK293 cells expressing GLAST-1 with and without PMA treatment. These data suggest that the neurotransmitter transporter activity of GLAST-1 is inhibited by phosphorylation at a non-PKC consensus site.     

Human T cells recovered from human/Balb radiation chimeras are hypersensitive to human immunodeficiency virus type 1 infection.

Replication of human immunodeficiency virus type 1 (HIV-1) is regulated by virus-encoded regulatory proteins, as well as by a variety of cellular factors. Productive infection of human T lymphocytes by HIV-1 is dependent upon the activation status of the target cells. In general, short-term mitogenic stimulation of CD4 T cells is used to enhance infection of peripheral blood mononuclear cells (PBMC) in vitro. Recently, we demonstrated that adoptive transfer of human PBMC into lethally irradiated BALB/c mice, radioprotected with severe combined immunodeficiency (SCID) mouse bone marrow, leads to marked T-cell activation and proliferation. In the present study, we investigated the effect of such xenoactivation of human T cells on their susceptibility to HIV-1 infection. Human cells that were recovered from human/Balb radiation chimeras supported efficient replication of laboratory strains of HIV-1, as well as of HIV-1 clinical isolates. The multiplicity of infection required to attain effective virus replication in the recovered xenoactivated human cells was 10- to 100-fold lower than that needed for infection of short- or long-term phytohemagglutinin (PHA)-stimulated blasts or of various T-cell lines. Analysis of human cell surface activation markers has indicated that xenoactivation in the mouse, in contrast to in vitro stimulation with PHA, is associated with a marked downregulation of CD25 (interleukin 2 receptor). Our results demonstrate that human cells recovered from human/Balb radiation chimeras, which are hypersensitive to HIV-1 infection, differ from in vitro-stimulated cells in their activation status. Therefore, this system could be used to study host factors that participate in HIV-1 infection and replication in vitro and in vivo.     

Pathways of glycosphingolipid biosynthesis in SW13 cells in the presence and absence of vimentin intermediate filaments

We reported previously that the incorporation of sugars intoglycosphingolipids (GSL) is diminished in SW13 cells that lacka vimentin intermediate filament (IF) network (vim–) comparedto vim+ cells. To further analyze the nature of this abnormality,we double-labeled cells with ³H-serine and ^l4C-sugars. Therewas no difference between vim+ and vim– cells in the incorporationof serine into GSL, although the usual difference in sugar incorporationwas observed. This indicated that the defect in vim– cellswas not in the incorporation of sugars into ceramide synthesizedde novo by acylation of sphinganine (pathway 1). Sugars canalso be incorporated into ceramide synthesized from sphingosinethat is derived from catabolism of sphin-golipids (pathway 2),and into GSL that recycle through the Golgi apparatus from endosomes(pathway 3). The amount of galactose and glucosamine incorporatedinto GSL in these three pathways was analyzed by the use oftwo inhibitors of sphingolipid biosynthesis. ß-Chloroala-nineinhibits the de novo synthesis of sphinganine (pathway 1), andfumonisin Bl inhibits the acylation of sphinganine and sphingosine(pathways 1 and 2). We were surprised to observe that in bothvim+ and vim– cells only 20–40% of sugar incorporationinto GSL took place in pathway 1, and 60–80% of sugarincorporation took place in the recycling pathways. Moreover,in contrast to larger GSL, GlcCer was not synthesized in pathway3. Our observations indicate that vimentin IF facilitate therecycling of GSL and sphingosine, and that the differences betweenvim+ and vim– cells are predominantly in pathways 2 and3. Furthermore, although it is generally believed that virtuallyall GSL are synthesized in the de novo pathway, these data indicatethat the recyling pathways predominate in the incorporationof sugars into GSL in SW13 cells. glycosphingolipid biosynthesis intermediate filaments SW13 cells sphingolipid recycling vimentin