Microbial Diversity and Function in Shallow Subsurface Sediment and Oceanic Lithosphere of the Atlantis Massif.

The marine lithospheric subsurface is one of the largest biospheres on Earth; however, little is known about the identity and ecological function of microorganisms found in low abundance in this habitat, though these organisms impact global-scale biogeochemical cycling. Here, we describe the diversity and metabolic potential of sediment and endolithic (within rock) microbial communities found in ultrasmall amounts (101 to 104 cells cm-3) in the subsurface of the Atlantis Massif, an oceanic core complex on the Mid-Atlantic Ridge that was sampled on International Ocean Discovery Program (IODP) Expedition 357. This study used fluorescence-activated cell sorting (FACS) to enable the first amplicon, metagenomic, and single-cell genomic study of the shallow (<20 m below seafloor) subsurface of an actively serpentinizing marine system. The shallow subsurface biosphere of the Atlantis Massif was found to be distinct from communities observed in the nearby Lost City alkaline hydrothermal fluids and chimneys, yet similar to other low-temperature, aerobic subsurface settings. Genes associated with autotrophy were rare, although heterotrophy and aerobic carbon monoxide and formate cycling metabolisms were identified. Overall, this study reveals that the shallow subsurface of an oceanic core complex hosts a biosphere that is not fueled by active serpentinization reactions and by-products. IMPORTANCE The subsurface rock beneath the ocean is one of the largest biospheres on Earth, and microorganisms within influence global-scale nutrient cycles. This biosphere is difficult to study, in part due to the low concentrations of microorganisms that inhabit the vast volume of the marine lithosphere. In spite of the global significance of this biosphere, little is currently known about the microbial ecology of such rock-associated microorganisms. This study describes the identity and genomic potential of microorganisms in the subsurface rock and sediment at the Atlantis Massif, an underwater mountain near the Mid-Atlantic Ridge. To enable our analyses, fluorescence-activated cell sorting (FACS) was used as a means to concentrate cells from low biomass environmental samples for genomic analyses. We found distinct rock-associated microorganisms and found that the capacity for microorganisms to utilize organic carbon was the most prevalent form of carbon cycling. We additionally identified a potential role for carbon monoxide metabolism in the subsurface.

Nucleic acid sequence bank.

In the report by T. Kakunaga and J. D. Crow (25 July, p. 505), Fig. 1 on page 506 should have been printed as follows: [See figure in the PDF file]

Protein Information Resource: a community resource for expert annotation of protein data.

The Protein Information Resource, in collaboration with the Munich Information Center for Protein Sequences (MIPS) and the Japan International Protein Information Database (JIPID), produces the most comprehensive and expertly annotated protein sequence database in the public domain, the PIR-International Protein Sequence Database. To provide timely and high quality annotation and promote database interoperability, the PIR-International employs rule-based and classification-driven procedures based on controlled vocabulary and standard nomenclature and includes status tags to distinguish experimentally determined from predicted protein features. The database contains about 200,000 non-redundant protein sequences, which are classified into families and superfamilies and their domains and motifs identified. Entries are extensively cross-referenced to other sequence, classification, genome, structure and activity databases. The PIR web site features search engines that use sequence similarity and database annotation to facilitate the analysis and functional identification of proteins. The PIR-Inter-national databases and search tools are accessible on the PIR web site at http://pir.georgetown.edu/ and at the MIPS web site at http://www.mips.biochem.mpg.de. The PIR-International Protein Sequence Database and other files are also available by FTP.

Flexibility of an active center in sodium-plus-potassium adenosine triphosphatase.

In plasma membranes of intact cells an enzymatic pump actively transports sodium ions inward and potassium ions outward. In preparations of broken membranes it appears as an adenosine triphosphatase dependent on magnesium, sodium, and potassium ions together. In this adenosine triphosphatase a phosphorylated intermediate is formed from adenosine triphosphate in the presence of sodium ions and is hydrolyzed with the addition of potassium ions. The normal intermediate was not split by adenosine diphosphate. However, selective poisoning by N-ethylmaleimide or partial inhibition by a low magnesium ion concentration yielded an intermediate split by adenosine diphosphate and insensitive to potassium ions. Pulse experiments on the native enzyme supported further a hypothesis of a sequence of phosphorylated forms, the first being made reversibly from adenosine triphosphate in the presence of sodium ion and the second being made irreversiblyfrom the first and hydrolyzed in the presence of potassium ion. The cardioactive steriod inhibitor, ouabain, appeared to combine preferentially with the second form. Phosphorylation was at the same active site according to electrophoretic patterns of proteolytic phosphorylated fragments of both reactive forms. It is concluded that there is a conformational change in the active center for phosphorylation during the normal reaction sequence. This change may be linked to one required theoretically for active translocation of ions across the cell membrane.

Ecological genomics of marine Roseobacters.

Bacterioplankton of the marine Roseobacter clade have genomes that reflect a dynamic environment and diverse interactions with marine plankton. Comparative genome sequence analysis of three cultured representatives suggests that cellular requirements for nitrogen are largely provided by regenerated ammonium and organic compounds (polyamines, allophanate, and urea), while typical sources of carbon include amino acids, glyoxylate, and aromatic metabolites. An unexpectedly large number of genes are predicted to encode proteins involved in the production, degradation, and efflux of toxins and metabolites. A mechanism likely involved in cell-to-cell DNA or protein transfer was also discovered: vir-related genes encoding a type IV secretion system typical of bacterial pathogens. These suggest a potential for interacting with neighboring cells and impacting the routing of organic matter into the microbial loop. Genes shared among the three roseobacters and also common in nine draft Roseobacter genomes include those for carbon monoxide oxidation, dimethylsulfoniopropionate demethylation, and aromatic compound degradation. Genes shared with other cultured marine bacteria include those for utilizing sodium gradients, transport and metabolism of sulfate, and osmoregulation.

Methods for identifying proteins by using partial sequences.

Methods for the identification of a protein segment by using the information from partial sequence analyses are described. If the protein sequence is known, a segment can usually be identified with confidence through comparison with the data file of all known sequences when the identity and position of only seven amino acid residues (not necessarily contiguous) are known. Partial sequences are obtained from extremely sensitive microsequencing procedures. Tissue is incubated with amino acids, one or more of which are distinctively radiolabeled. Proteins of interest are isolated and a sequenator experiment performed to locate the positions of radioactivity in an NH(2)-terminal segment of approximately 30 residues. We derive and investigate an equation for the probability of finding a unique match to any pattern of radioactivity. From this we suggest a new strategy. In one incubation, several amino acids are labeled with each kind of isotope. The most information is contained in patterns in which approximately equal numbers of positions are occupied by residues distinguished by different labels (including no label). The amino acid composition of the segment will typically not be known in advance. Labeling residues expected to occupy 36% of the positions suffices for a 98% chance of success in uniquely characterizing any human segment. Such a strategy will permit the identification of most proteins from a single tissue incubation. The mathematical discussion is general and applies to any segment from a sequence and to sequences obtained by any method. Improved identification procedures should expedite the accumulation of information on the expression and function of proteins.

Comparison of histologic and functional recovery after peripheral nerve repair.

After repair, nerve stains of skin and subcutaneous tissue biopsies of 23 fingertips contained identifiable axons in all patients. The level of recovery was more advanced in some patients than in others, as manifested by identifiable nerve in various plexuses and receptors. Meissner's corpuscles were observed to be reinnervated in 12 of 17 patients and in 16 of 23 fingertips. The degree and level of reinnervation did not correlate with clinical testing or subjective impression of the result. The authors conclude that sensibility is the result of several factors active in the peripheral and central nervous system. Axonal regrowth is but one of these.

PIR-ALN: a database of protein sequence alignments.

MOTIVATION: The Protein Information Resource (PIR) maintains a database of annotated and curated alignments in order to visually represent interrelationships among sequences in the PIR-International Protein Sequence Database, to spread and standardize protein names, features and keywords among members of a family or superfamily, and to aid us in classifying sequences, in identifying conserved regions, and in defining new homology domains. RESULTS: Release 22.0, (December 1998), of the PIR-ALN database contains a total of 3806 alignments, including 1303 superfamily, 2131 family and 372 homology domain alignments. This is an appropriate dataset to develop and extract patterns, test profiles, train neural networks or build Hidden Markov Models (HMMs). These alignments can be used to standardize and spread annotation to newer members by homology, as well as to understand the modular architecture of multidomain proteins. PIR-ALN includes 529 alignments that can be used to develop patterns not represented in PROSITE, Blocks, PRINTS and Pfam databases. The ATLAS information retrieval system can be used to browse and query the PIR-ALN alignments. AVAILABILITY: PIR-ALN is currently being distributed as a single ASCII text file along with the title, member, species, superfamily and keyword indexes. The quarterly and weekly updates can be accessed via the WWW at pir.georgetown.edu. The quarterly updates can also be obtained by anonymous FTP from the PIR FTP site at NBRF.Georgetown.edu, directory [ANONYMOUS.PIR.ALIGNMENT].

Database of protein sequence alignments: PIR-ALN.

The Protein Information Resource (PIR) has been maintaining a database of curated protein sequence alignments since 1991. The collection includes superfamily, family and homology domain alignments. CLUSTAL V/W is used to generate multiple sequence alignments and ALNED, an interactive alignment editor, is used to check and correct them. The database has helped in classifying sequences, in defining new homology domains, and in spreading and standardizing protein names, features and keywords among members of a family or superfamily. The ATLAS information retrieval system can be used to browse and query the PIR-ALN alignments. The quarterly and weekly updates can be accessed via the WWW at http://www-nbrf. georgetown.edu/pir/

PIR: a new resource for bioinformatics.

UNLABELLED: The Protein Information Resource (PIR) has greatly expanded its Web site and developed a set of interactive search and analysis tools to facilitate the analysis, annotation, and functional identification of proteins. New search engines have been implemented to combine sequence similarity search results with database annotation information. The new PIR search systems have proved very useful in providing enriched functional annotation of protein sequences, determining protein superfamily-domain relationships, and detecting annotation errors in genomic database archives. AVAILABILITY: http://pir.georgetown.edu/. CONTACT: mcgarvey@nbrf.georgetown.edu

The PIR-International Protein Sequence Database.

The Protein Information Resource (PIR; http://www-nbrf.georgetown. edu/pir/) supports research on molecular evolution, functional genomics, and computational biology by maintaining a comprehensive, non-redundant, well-organized and freely available protein sequence database. Since 1988 the database has been maintained collaboratively by PIR-International, an international association of data collection centers cooperating to develop this resource during a period of explosive growth in new sequence data and new computer technologies. The PIR Protein Sequence Database entries are classified into superfamilies, families and homology domains, for which sequence alignments are available. Full-scale family classification supports comparative genomics research, aids sequence annotation, assists database organization and improves database integrity. The PIR WWW server supports direct on-line sequence similarity searches, information retrieval, and knowledge discovery by providing the Protein Sequence Database and other supplementary databases. Sequence entries are extensively cross-referenced and hypertext-linked to major nucleic acid, literature, genome, structure, sequence alignment and family databases. The weekly release of the Protein Sequence Database can be accessed through the PIR Web site. The quarterly release of the database is freely available from our anonymous FTP server and is also available on CD-ROM with the accompanying ATLAS database search program.

The PIR-International Protein Sequence Database.

From its origin the Protein Information Resource (http://www-nbrf. georgetown.edu/pir/) has supported research on evolution and computational biology by designing and compiling a comprehensive, quality controlled, and well-organized protein sequence database. The database has been produced and updated on a regular schedule since 1984. Since 1988 it has been maintained collaboratively by the PIR-International, an association of data collection centers engaged in international cooperation for the development of this research resource during a period of explosive acquisition of new data. As of June 1997, essentially all sequence entries have been classified into families, allowing the efficient application of methods to propagate and standardize annotation among related sequences. The databases are available through the Internet by the World-Wide Web and FTP, or on CD-ROM and magnetic media.

Asphalt volcanism and chemosynthetic life in the Campeche Knolls, Gulf of Mexico.

In the Campeche Knolls, in the southern Gulf of Mexico, lava-like flows of solidified asphalt cover more than 1 square kilometer of the rim of a dissected salt dome at a depth of 3000 meters below sea level. Chemosynthetic tubeworms and bivalves colonize the sea floor near the asphalt, which chilled and contracted after discharge. The site also includes oil seeps, gas hydrate deposits, locally anoxic sediments, and slabs of authigenic carbonate. Asphalt volcanism creates a habitat for chemosynthetic life that may be widespread at great depth in the Gulf of Mexico.

The Protein Information Resource (PIR) and the PIR-International Protein Sequence Database.

From its origin, the PIR has aspired to support research in computational biology and genomics through the compilation of a comprehensive, quality controlled and well-organized protein sequence information resource. The resource originated with the pioneering work of the late Margaret O. Dayhoff in the early 1960s. Since 1988, the Protein Sequence Database has been maintained collaboratively by PIR-International, an association of macromolecular sequence data collection centers dedicated to fostering international cooperation as an essential element in the development of scientific databases. The work of the resource is widely distributed and is available on the World Wide Web, via FTP, E-mail server, CD-ROM and magnetic media. It is widely redistributed and incorporated into many other protein sequence data compilations including SWISS-PROT and theEntrezsystem of the NCBI.

The protein information resource (PIR).

The Protein Information Resource (PIR) produces the largest, most comprehensive, annotated protein sequence database in the public domain, the PIR-International Protein Sequence Database, in collaboration with the Munich Information Center for Protein Sequences (MIPS) and the Japan International Protein Sequence Database (JIPID). The expanded PIR WWW site allows sequence similarity and text searching of the Protein Sequence Database and auxiliary databases. Several new web-based search engines combine searches of sequence similarity and database annotation to facilitate the analysis and functional identification of proteins. New capabilities for searching the PIR sequence databases include annotation-sorted search, domain search, combined global and domain search, and interactive text searches. The PIR-International databases and search tools are accessible on the PIR WWW site at http://pir.georgetown.edu and at the MIPS WWW site at http://www. mips.biochem.mpg.de. The PIR-International Protein Sequence Database and other files are also available by FTP.