The evolution of the marine carbonate factory.

Calcium carbonate formation is the primary pathway by which carbon is returned from the ocean-atmosphere system to the solid Earth1,2. The removal of dissolved inorganic carbon from seawater by precipitation of carbonate minerals-the marine carbonate factory-plays a critical role in shaping marine biogeochemical cycling1,2. A paucity of empirical constraints has led to widely divergent views on how the marine carbonate factory has changed over time3-5. Here we use geochemical insights from stable strontium isotopes to provide a new perspective on the evolution of the marine carbonate factory and carbonate mineral saturation states. Although the production of carbonates in the surface ocean and in shallow seafloor settings have been widely considered the predominant carbonate sinks for most of the history of the Earth6, we propose that alternative processes-such as porewater production of authigenic carbonates-may have represented a major carbonate sink throughout the Precambrian. Our results also suggest that the rise of the skeletal carbonate factory decreased seawater carbonate saturation states.

Isotope systematics of subfossil, historical, and modern Nautilus macromphalus from New Caledonia.

Cephalopod carbonate geochemistry underpins studies ranging from Phanerozoic, global-scale change to outcrop-scale paleoecological reconstructions. Interpreting these data hinges on assumed similarity to model organisms, such as Nautilus, and generalization from other molluscan biomineralization processes. Aquarium rearing and capture of wild Nautilus suggest shell carbonate precipitates quickly (35 µm/day) in oxygen isotope equilibrium with seawater. Other components of Nautilus shell chemistry are less well-studied but have potential to serve as proxies for paleobiology and paleoceanography. To calibrate the geochemical response of cephalopod δ15Norg, δ13Corg, δ13Ccarb, δ18Ocarb, and δ44/40Cacarb to modern anthropogenic environmental change, we analyzed modern, historical, and subfossil Nautilus macromphalus from New Caledonia. Samples span initial human habitation, colonialization, and industrial pCO2 increase. This sampling strategy is advantageous because it avoids the shock response that can affect geochemical change in aquarium experiments. Given the range of living depths and more complex ecology of Nautilus, however, some anthropogenic signals, such as ocean acidification, may not have propagated to their living depths. Our data suggest some environmental changes are more easily preserved than others given variability in cephalopod average living depth. Calculation of the percent respired carbon incorporated into the shell using δ13Corg, δ13Ccarb, and Suess-effect corrected δ13CDIC suggests an increase in the last 130 years that may have been caused by increasing carbon dioxide concentration or decreasing oxygen concentration at the depths these individuals inhabited. This pattern is consistent with increasing atmospheric CO2 and/or eutrophication offshore of New Caledonia. We find that δ44/40Ca remains stable across the last 130 years. The subfossil shell from a cenote may exhibit early δ44/40Ca diagenesis. Questions remain about the proportion of dietary vs ambient seawater calcium incorporation into the Nautilus shell. Values of δ15N do not indicate trophic level change in the last 130 years, and the subfossil shell may show diagenetic alteration of δ15N toward lower values. Future work using historical collections of Sepia and Spirula may provide additional calibration of fossil cephalopod geochemistry.

Technical Note: Calcium and carbon stable isotope ratios as paleodietary indicators.

Calcium stable isotope ratios are hypothesized to vary as a function of trophic level. This premise raises the possibility of using calcium stable isotope ratios to study the dietary behaviors of fossil taxa and to test competing hypotheses on the adaptive origins of euprimates. To explore this concept, we measured the stable isotope composition of contemporary mammals in northern Borneo and northwestern Costa Rica, two communities with functional or phylogenetic relevance to primate origins. We found that bone collagen δ(13) C and δ(15) N values could differentiate trophic levels in each assemblage, a result that justifies the use of these systems to test the predicted inverse relationship between bioapatite δ(13) C and δ(44) Ca values. As expected, taxonomic carnivores (felids) showed a combination of high δ(13) C and low δ(44) Ca values; however, the δ(44) Ca values of other faunivores were indistinguishable from those of primary consumers. We suggest that the trophic insensitivity of most bioapatite δ(44) Ca values is attributable to the negligible calcium content of arthropod prey. Although the present results are inconclusive, the tandem analysis of δ(44) Ca and δ(13) C values in fossils continues to hold promise for informing paleodietary studies and we highlight this potential by drawing attention to the stable isotope composition of the Early Eocene primate Cantius.

Autoregulation of the 26S proteasome by in situ ubiquitination.

The 26S proteasome degrades ubiquitinated proteins, and proteasomal degradation controls various cellular events. Here we report that the human 26S proteasome is ubiquitinated, by which the ubiquitin receptors Adrm1 and S5a, the ATPase subunit Rpt5, and the deubiquitinating enzyme Uch37 are ubiquitinated in situ by proteasome-associating ubiquitination enzymes. Ubiquitination of these subunits significantly impairs the 26S proteasome's ability to bind, deubiquitinate, and degrade ubiquitinated proteins. Moreover, ubiquitination of the 26S proteasome can be antagonized by proteasome-residing deubiquitinating enzymes, by the binding of polyubiquitin chains, and by certain cellular stress, indicating that proteasome ubiquitination is dynamic and regulated in cells. We propose that in situ ubiquitination of the 26S proteasome regulates its activity, which could function to adjust proteasomal activity in response to the alteration of cellular ubiquitination levels.

Functions of the 19S complex in proteasomal degradation.

The 26S proteasome degrades ubiquitylated proteins. It consists of the 20S proteasome and the PA700/19S complex. PA700 plays essential roles in processing ubiquitylated substrates; it can bind, deubiquitylate, and unfold ubiquitylated proteins, which then translocate into the proteolytic chamber of the 20S proteasome for degradation. Here, we summarize the current knowledge of PA700-mediated substrate binding and deubiquitylation, and provide models to explain how substrate binding and deubiquitylation could regulate proteasomal degradation. We also discuss the features and potential therapeutic uses of the two recently identified small molecule inhibitors of the proteasome-residing deubiquitylating enzymes.

Ubiquitin chain trimming recycles the substrate binding sites of the 26 S proteasome and promotes degradation of lysine 48-linked polyubiquitin conjugates.

The 26 S proteasome possesses two distinct deubiquitinating activities. The ubiquitin (Ub) chain amputation activity removes the entire polyUb chain from the substrates. The Ub chain trimming activity progressively cleaves a polyUb chain from the distal end. The Ub chain amputation activity mediates degradation-coupled deubiquitination. The Ub chain trimming activity can play a supportive or an inhibitory role in degradation, likely depending on features of the substrates. How Ub chain trimming assists degradation is not clear. We find that inhibition of the chain trimming activity of the 26 S proteasome with Ub aldehyde significantly inhibits degradation of Ub4 (Lys-48)-UbcH10 and causes accumulation of free Ub4 (generated from chain amputation) that can be retained on the proteasome. Also, a non-trimmable Lys-48-mimic Ub4 efficiently targets UbcH10 to the 26 S proteasome, but it cannot support efficient degradation of UbcH10 compared with regular Lys-48 Ub4. These results indicate that polyUb chain trimming promotes proteasomal degradation of Lys-48-linked substrates. Mechanistically, we propose that Ub chain trimming cleaves the proteasome-bound Lys-48-linked polyUb chains, which vacates the Ub binding sites of the 26 S proteasome, thus allowing continuous substrate loading.

The lysine 48 and lysine 63 ubiquitin conjugates are processed differently by the 26 s proteasome.

The role of Lys-63 ubiquitin chains in targeting proteins for proteasomal degradation is still obscure. We systematically compared proteasomal processing of Lys-63 ubiquitin chains with that of the canonical proteolytic signal, Lys-48 ubiquitin chains. Quantitative mass spectrometric analysis of ubiquitin chains in HeLa cells determines that the levels of Lys-63 ubiquitin chains are insensitive to short-time proteasome inhibition. Also, the Lys-48/Lys-63 ratio in the 26 S proteasome-bound fraction is 1.7-fold more than that in the cell lysates, likely because some cellular Lys-63 ubiquitin conjugates are sequestered by Lys-63 chain-specific binding proteins. In vitro, Lys-48 and Lys-63 ubiquitin chains bind the 26 S proteasome comparably, whereas Lys-63 chains are deubiquitinated 6-fold faster than Lys-48 chains. Also, Lys-63 tetraubiquitin-conjugated UbcH10 is rapidly deubiquitinated into the monoubiquitinated form, whereas Lys-48 tetraubiquitin targets UbcH10 for degradation. Furthermore, we found that both the ubiquitin aldehyde- and 1,10-phenanthroline-sensitive deubiquitinating activities of the 26 S proteasome contribute to Lys-48- and Lys-63-linkage deubiquitination, albeit the inhibitory extents are different. Together, our findings suggest that compared with Lys-48 chains, cellular Lys-63 chains have less proteasomal accessibility, and proteasome-bound Lys-63 chains are more rapidly deubiquitinated, which could cause inefficient degradation of Lys-63 conjugates.

Bacteriophage-based vectors for site-specific insertion of DNA in the chromosome of Corynebacteria.

In Corynebacterium diphtheriae, diphtheria toxin is encoded by the tox gene of some temperate corynephages such as beta. beta-like corynephages are capable of inserting into the C. diphtheriae chromosome at two specific sites, attB1 and attB2. Transcription of the phage-encoded tox gene, and many chromosomally encoded genes, is regulated by the DtxR protein in response to Fe(2+) levels. Characterizing DtxR-dependent gene regulation is pivotal in understanding diphtheria pathogenesis and mechanisms of iron-dependent gene expression; although this has been hampered by a lack of molecular genetic tools in C. diphtheriae and related Coryneform species. To expand the systems for genetic manipulation of C. diphtheriae, we constructed plasmid vectors capable of integrating into the chromosome. These plasmids contain the beta-encoded attP site and the DIP0182 integrase gene of C. diphtheriae NCTC13129. When these vectors were delivered to the cytoplasm of non-lysogenic C. diphtheriae, they integrated into either the attB1 or attB2 sites with comparable frequency. Lysogens were also transformed with these vectors, by virtue of the second attB site. An integrated vector carrying an intact dtxR gene complemented the mutant phenotypes of a C. diphtheriae DeltadtxR strain. Additionally, strains of beta-susceptible C. ulcerans, and C. glutamicum, a species non-permissive for beta, were each transformed with these vectors. This work significantly extends the tools available for targeted transformation of both pathogenic and non-pathogenic Corynebacterium species.

Transcription of the contiguous sigB, dtxR, and galE genes in Corynebacterium diphtheriae: evidence for multiple transcripts and regulation by environmental factors.

The iron-dependent transcriptional regulator DtxR from Corynebacterium diphtheriae is the prototype for a family of metal-dependent regulators found in diverse bacterial species. The structure of DtxR and its action as a repressor have been extensively characterized, but little is known about expression of dtxR. In the current study, we investigated transcription of dtxR as well as the sigB and galE genes located immediately upstream and downstream from dtxR, respectively. We identified two promoters that determine transcription of dtxR. The first, located upstream of sigB, appears to be controlled by an extracytoplasmic function sigma factor. The second, located in the intergenic region between sigB and dtxR, is similar to promoters used by the primary vegetative sigma factors in other actinomycete species. Using quantitative real-time assays, we demonstrated that the number of transcripts initiated upstream from sigB is affected by several environmental factors. In contrast, the presence of sodium dodecyl sulfate was the only factor tested that conclusively affects the number of transcripts initiated in the sigB-dtxR intergenic region. Additionally, we provided evidence for the existence of transcripts that contain sigB, dtxR, and galE. Our studies provide the first quantitative transcriptional analysis of a gene encoding a DtxR family regulator and give new insights into transcriptional regulation in C. diphtheriae.