Author Correction: Forearc carbon sink reduces long-term volatile recycling into the mantle.

An Amendment to this paper has been published and can be accessed via a link at the top of the paper.

Author Correction: Forearc carbon sink reduces long-term volatile recycling into the mantle.

Change history: In this Article, the original affiliation 2 was not applicable and has been removed. In addition, in the Acknowledgements there was a statement missing and an error in a name. These errors have been corrected online.

Forearc carbon sink reduces long-term volatile recycling into the mantle.

Carbon and other volatiles in the form of gases, fluids or mineral phases are transported from Earth's surface into the mantle at convergent margins, where the oceanic crust subducts beneath the continental crust. The efficiency of this transfer has profound implications for the nature and scale of geochemical heterogeneities in Earth's deep mantle and shallow crustal reservoirs, as well as Earth's oxidation state. However, the proportions of volatiles released from the forearc and backarc are not well constrained compared to fluxes from the volcanic arc front. Here we use helium and carbon isotope data from deeply sourced springs along two cross-arc transects to show that about 91 per cent of carbon released from the slab and mantle beneath the Costa Rican forearc is sequestered within the crust by calcite deposition. Around an additional three per cent is incorporated into the biomass through microbial chemolithoautotrophy, whereby microbes assimilate inorganic carbon into biomass. We estimate that between 1.2 × 108 and 1.3 × 1010 moles of carbon dioxide per year are released from the slab beneath the forearc, and thus up to about 19 per cent less carbon is being transferred into Earth's deep mantle than previously estimated.

Thiosulfate oxidation by Thiomicrospira thermophila: metabolic flexibility in response to ambient geochemistry.

Previous studies of the stoichiometry of thiosulfate oxidation by colorless sulfur bacteria have failed to demonstrate mass balance of sulfur, indicating that unidentified oxidized products must be present. Here the reaction stoichiometry and kinetics under variable pH conditions during the growth of Thiomicrospira thermophila strain EPR85, isolated from diffuse hydrothermal fluids at the East Pacific Rise, is presented. At pH 8.0, thiosulfate was stoichiometrically converted to sulfate. At lower pH, the products of thiosulfate oxidation were extracellular elemental sulfur and sulfate. We were able to replicate previous experiments and identify the missing sulfur as tetrathionate, consistent with previous reports of the activity of thiosulfate dehydrogenase. Tetrathionate was formed under slightly acidic conditions. Genomic DNA from T. thermophila strain EPR85 contains genes homologous to those in the Sox pathway (soxAXYZBCDL), as well as rhodanese and thiosulfate dehydrogenase. No other sulfur oxidizing bacteria containing sox(CD)2 genes have been reported to produce extracellular elemental sulfur. If the apparent modified Sox pathway we observed in T. thermophila is present in marine Thiobacillus and Thiomicrospira species, production of extracellular elemental sulfur may be biogeochemically important in marine sulfur cycling.

Contribution of aerobic photoheterotrophic bacteria to the carbon cycle in the ocean.

The vertical distribution of bacteriochlorophyll a, the numbers of infrared fluorescent cells, and the variable fluorescence signal at 880 nanometers wavelength, all indicate that photosynthetically competent anoxygenic phototrophic bacteria are abundant in the upper open ocean and comprise at least 11% of the total microbial community. These organisms are facultative photoheterotrophs, metabolizing organic carbon when available, but are capable of photosynthetic light utilization when organic carbon is scarce. They are globally distributed in the euphotic zone and represent a hitherto unrecognized component of the marine microbial community that appears to be critical to the cycling of both organic and inorganic carbon in the ocean.

A family of Shc related proteins with conserved PTB, CH1 and SH2 regions.

Shc proteins are targets of activated tyrosine kinases and have been implicated in the transmission of activation signals to Ras. Upon phosphorylation, Shc proteins form stable complexes with cellular tyrosine-phosphorylated proteins and with the Grb2 adaptor protein. Two Shc isoforms of 52 and 46 kDa have been characterized. They share a C-terminal SH2 domain, a proline- and glycine-rich region (collagen homologous region 1; CH1) and a N-terminal phospho-tyrosine binding domain (PTB). We report her ethe initial characterization of two Shc related human cDNAs: ShcB and ShcC. The ShcB and ShcC cDNAs code for proteins that are highly similar and share the same modular organization as Shc. PTB and SH2 domains of ShcB and ShcC have similar binding specificities in vitro and bind to activated EGFR in a phosphotyrosine-dependent manner. Based on these findings we propose to rename Shc as ShcA. Anti-ShcB and anti-ShcC antibodies recognize specific polypeptides of 52, 47 kDa (ShcB) and 54 kDa (ShcC) in mammalian cells. Since these two genes are predominantly expressed in specific brain tissues, these Shc family members may be involved in cell type-specific signaling, in the nervous system.

Linking an easily detectable phenotype to the folding of a common structural motif. Selection of rare turn mutations that prevent the folding of Rop.

Rop is the simplest and most regular member of a family of proteins characterized by a bundle of four antiparallel helices. Rop is dimeric, each monomer being formed by two helices connected by a sharp bend. In this work we have extensively mutagenized three residues that form the connection between the two alpha-helices to ask whether the bend region contains any important folding information. The characterization of a collection of random mutants indicated that this structure is rather insensitive to amino acid substitutions and that most amino acids are tolerated in these positions by the Rop native structure. In order to identify the rare amino acid sequences that would prevent Rop from folding and/or dimerizing, we exploited the observation that Rop can functionally substitute the dimerization domain of the lambda repressor. In fact plasmids expressing a hybrid protein formed by the amino-terminal domain of the lambda repressor covalently linked to Rop, confer immunity to lambda infection on their hosts. We have shown that this property depends on the ability of the Rop moiety to fold and dimerize. The analysis of 380 Rop mutants containing random amino acid sequences at positions 30, 31 and 32 allowed us to identify three mutant Rop proteins that are defective in dimerization, probably as a consequence of their inability to fold. In these mutants the tripeptides VED, VPD and YPD substitute the wild-type DAD at positions 30, 31 and 32. Other combinations of amino acids are found resulting in levels of immunity that are lower than the wild-type but still sufficient to prevent single plaque formation. This result suggests that a smaller proportion of the corresponding Rop protein reaches a thermodynamic and proteolytically stable dimeric state.

Modified phage peptide libraries as a tool to study specificity of phosphorylation and recognition of tyrosine containing peptides.

Tyrosine phosphorylation and protein recognition, mediated by phosphotyrosine containing peptides, play an important role in determining the specific response of a cell, when stimulated by external signals. We have used peptide repertoires displayed by filamentous phage as a tool to study the substrate specificity of the protein tyrosine kinase (PTK) p55(fyn) (Fyn). Peptide libraries were incubated for a short time in the presence of Fyn and phages displaying efficiently phosphorylated peptides were selected by panning over anti-phosphotyrosine antibodies. The characterization of the peptides enriched after three phosphorylation/selection rounds allowed us to define a canonical substrate sequence for the kinase Fyn, E-(phi/T)YGx phi, where phi represents any hydrophobic residue. A peptide conforming to this sequence is a better substrate than a second peptide designed to be in accord with the consensus sequence recognised by the Fyn SH2 domain. When the library phosphorylation reaction is carried out in saturation conditions, practically all the tyrosine containing peptides are phosphorylated, irrespective of their context. These "fully modified" peptide libraries are a valuable tool to study the specificity of phosphotyrosine mediated protein recognition. We have used this new tool to identify a family of peptides that bind the PTB domain of the adapter protein Shc. Comparison of the peptide sequences permits us to confirm the essential role of N at position -3, while P often found at position -2 in natural targets is not absolutely required. Furthermore, our approach permits us to reveal an "extended" consensus indicating that residues that do not seem to influence binding in natural peptides can make productive contacts, at least in linear peptides.

Structure determination of the glutamate dehydrogenase from the hyperthermophile Thermococcus litoralis and its comparison with that from Pyrococcus furiosus.

Glutamate dehydrogenase catalyses the oxidative deamination of glutamate to 2-oxoglutarate with concomitant reduction of NAD(P)(+), and has been shown to be widely distributed in nature across species ranging from psychrophiles to hyperthermophiles. Extensive characterisation of this enzyme isolated from hyperthermophilic organisms has led to its adoption as a model system for analysing the determinants of thermal stability. The crystal structure of the extremely thermostable glutamate dehydrogenase from Thermococcus litoralis has been determined at 2.5 A resolution, and has been compared to that from the hyperthermophile Pyrococcus furiosus. The two enzymes are 87 % identical in sequence, yet differ 16-fold in their half-lives at 104 degrees C. This is the first reported comparative analysis of the structures of a multisubunit enzyme from two closely related yet distinct hyperthermophilies. The less stable T. litoralis enzyme has a decreased number of ion pair interactions; modified patterns of hydrogen bonding resulting from isosteric sequence changes; substitutions that decrease packing efficiency; and substitutions which give rise to subtle but distinct shifts in both main-chain and side-chain elements of the structure. This analysis provides a rational basis to test ideas on the factors that confer thermal stability in proteins through a combination of mutagenesis, calorimetry, and structural studies.

Pressure-induced thermostabilization of glutamate dehydrogenase from the hyperthermophile Pyrococcus furiosus.

In this paper, elevated pressures up to 750 atm (1 atm = 101 kPa) were found to have a strong stabilizing effect on two extremely thermophilic glutamate dehydrogenases (GDHs): the native enzyme from the hyperthermophile Pyrococcus furiosus (Pf), and a recombinant GDH mutant containing an extra tetrapeptide at the C-terminus (rGDHt). The presence of the tetrapeptide greatly destabilized the recombinant mutant at ambient pressure; however, the destabilizing effect was largely reversed by the application of pressure. Electron spin resonance (ESR) spectroscopy of a spin-label attached to the terminal cysteine of rGDHt revealed a high degree of mobility, suggesting that destabilization is due to weakened intersubunit ion-pair interactions induced by thermal fluctuations of the tetrapeptide. For both enzymes, the stabilizing effect of pressure increased with temperature as well as pressure, reaching 36-fold for rGDHt at 105 degrees C and 750 atm, the largest pressure-induced thermostabilization of an enzyme reported to date. Stabilization of both native GDH and rGDHt was also achieved by adding glycerol. Based on the kinetics of thermal inactivation and the known effects of glycerol on protein structure, a mechanism of pressure-induced thermostabilization is proposed.

Recovery and phylogenetic analysis of archaeal rRNA sequences from continental shelf sediments.

Phylogenetic analyses of archaeal 16S rRNA genes (rDNA) from DNA extracted from continental shelf sediments revealed the presence of two major lineages, belonging to the kingdoms Crenarchaeota and Euryarchacota, respectively. Our analyses indicate that the benthic Archaea belong to a new group, divergent from the marine low-temperature planktonic Archaea. This is the first report showing the existence of Archaea, unrelated to methanogens, specifically associated with low-temperature anoxic marine sediments.

Siderophore production by Salmonella species isolated from different sources.

A total of 230 Salmonella strains were screened for enterobactin and aerobactin production, sensitivity to bacteriocins and resistance to antibiotics. All the isolates produced the phenolate siderophore enterobactin. Amongst these, 74 strains, most belonging to S. enteritidis, were sensitive to colicin B. Only 26 isolates, all belonging to S. wien, produced an additional iron chelator, i.e. the siderophore aerobactin, and 22 out of these were sensitive to cloacin DF13. Analysis of iron repressible outer membrane proteins and plasmid profiles in S. wien strains showed that the expression of a 74-kDa iron-repressible outer membrane protein and the presence of large plasmids were associated with multiple antibiotic resistance, aerobactin production and sensitivity to cloacin DF13. The incidence of aerobactin-producing strains among S. wien isolates was higher during years 1974-1985; the epidemiological implications of these results are discussed.

Epidemiological typing of uropathogenic Pseudomonas aeruginosa strains from hospitalized patients.

One hundred and twenty-one clinical isolates of Pseudomonas aeruginosa from patients with hospital-acquired urinary tract infections (UTIs) were studied to determine their major epidemiological markers, including API 20NE profile, O-serotype, pyocin type, phage type, lysogenic state and antibiotic susceptibility. Serotypes O4, O12, O11, O6 and O5 were found with a high frequency, accounting respectively for 23.9%, 23.1%, 12.3%, 8.2% and 5.7% of isolates. Pyocin type 10 was most common (32.2%) followed by types 1 (10.7%), 33 (7.5%) and 105 (4.1%); subtype h was predominant being characteristic of 34.7% of isolates. Most of the strains (69.4%) were either not phage typable or sensitive to phages 68 and 119x. Resistance to gentamicin, tobramycin, amikacin, imipenem and ciprofloxacin was more frequent among strains belonging to serotype O12. The O-serotypes were combined with API 20NE profiles, pyocin and phage types, lysogenic states and antibiotic resistance in order to identify epidemiologically related clones. Within predominant serotypes--O4 and O12--most strains displayed similar but not identical type characteristics, whereas other serotypes were less homogeneous. Our results support the concept that a combination of current typing techniques allows the identification of epidemiologically related P. aeruginosa isolates.

Virulence determinants in Pseudomonas aeruginosa strains from urinary tract infections.

A total of 121 uropathogenic Pseudomonas aeruginosa strains were examined for production of several virulence-related factors. These strains were distributed in five predominant O-serotypes, i.e. O 4, O 12, O 11, O 6 and O 5, which accounted respectively for 23.9, 23.1, 12.3, 8.2 and 5.7% of isolates. Pyochelin and pyoverdin siderophores were produced by most of the isolates, defective variants occurring at very low frequency (2.4% for pyochelin and 7.4% for pyoverdin). Adherence to uroepithelial cells and production of cytotoxins was demonstrated in 52.8 and 67.7% of the strains, respectively, with higher frequencies for epidemiologically related strains belonging to serotypes O 4 and O 12. Titration of total proteases, elastase and phospholipase C revealed a high degree of heterogeneity among isolates. However, examination of individual O-serotypes by exoenzyme production showed that elevated levels of total proteases and elastase were characteristics of serotypes of minor numerical importance, i.e. O 1, O 10, O 11 and O 17, whilst low levels of elastase were produced by strains belonging to the predominant serotypes, namely O 4 and O 12. Moreover, epidemiologically related strains belonging to serotypes O 4 and O 12 appeared more homogeneous than the whole serogroup, when compared with other groups on the basis of exoenzyme levels.

Population structure and phylogenetic characterization of marine benthic Archaea in deep-sea sediments.

During the past few years Archaea have been recognized as a widespread and significant component of marine picoplankton assemblages and, more recently, the presence of novel archaeal phylogenetic lineages has been reported in coastal marine benthic environments. We investigated the relative abundance, vertical distribution, phylogenetic composition, and spatial variability of Archaea in deep-sea sediments collected from several stations in the Atlantic Ocean. Quantitative oligonucleotide hybridization experiments indicated that the relative abundance of archaeal 16S rRNA in deep-sea sediments (1500 m deep) ranged from about 2.5 to 8% of the total prokaryotic rRNA. Clone libraries of PCR-amplified archaeal rRNA genes (rDNA) were constructed from 10 depth intervals obtained from sediment cores collected at depths of 1,500, 2,600, and 4,500 m. Phylogenetic analysis of rDNA sequences revealed the presence of a complex archaeal population structure, whose members could be grouped into discrete phylogenetic lineages within the two kingdoms, Crenarchaeota and Euryarchaeota. Comparative denaturing gradient gel electrophoresis profile analysis of archaeal 16S rDNA V3 fragments revealed a significant depth-related variability in the composition of the archaeal population.

Protein thermostability above 100 degreesC: a key role for ionic interactions.

The discovery of hyperthermophilic microorganisms and the analysis of hyperthermostable enzymes has established the fact that multisubunit enzymes can survive for prolonged periods at temperatures above 100 degreesC. We have carried out homology-based modeling and direct structure comparison on the hexameric glutamate dehydrogenases from the hyperthermophiles Pyrococcus furiosus and Thermococcus litoralis whose optimal growth temperatures are 100 degreesC and 88 degreesC, respectively, to determine key stabilizing features. These enzymes, which are 87% homologous, differ 16-fold in thermal stability at 104 degreesC. We observed that an intersubunit ion-pair network was substantially reduced in the less stable enzyme from T. litoralis, and two residues were then altered to restore these interactions. The single mutations both had adverse effects on the thermostability of the protein. However, with both mutations in place, we observed a fourfold improvement of stability at 104 degreesC over the wild-type enzyme. The catalytic properties of the enzymes were unaffected by the mutations. These results suggest that extensive ion-pair networks may provide a general strategy for manipulating enzyme thermostability of multisubunit enzymes. However, this study emphasizes the importance of the exact local environment of a residue in determining its effects on stability.

Insights into the molecular basis of thermal stability from the analysis of ion-pair networks in the glutamate dehydrogenase family.

The recent structure determination of glutamate dehydrogenase from the hyperthermophile Pyrococcus furiosus and the comparison of this structure with its counterparts from the mesophiles Clostridium symbiosum and Escherichia coli has highlighted the formation of extended networks of ion-pairs as a possible explanation for the superior thermal stability of the hyperthermostable enzyme. In the light of this, we have carried out a homology-based modelling study using sequences of a range of glutamate dehydrogenases drawn from species which span a wide spectrum of optimal growth temperatures. We have attempted to analyse the extent of the formation of ion-pair networks in these different enzymes and tried to correlate this with the observed thermal stability. The results of this analysis indicate that the ion-pair networks become more fragmented as the temperature stability of the enzyme decreases and are consistent with a role for the involvement of such networks in the adaptation of enzymes to extreme temperatures.