Studies of deeply buried, sedimentary microbial communities and associated biogeochemical processes during Ocean Drilling Program Leg 201 showed elevated prokaryotic cell numbers in sediment layers where methane is consumed anaerobically at the expense of sulfate. Here, we show that extractable archaeal rRNA, selecting only for active community members in these ecosystems, is dominated by sequences of uncultivated Archaea affiliated with the Marine Benthic Group B and the Miscellaneous Crenarchaeotal Group, whereas known methanotrophic Archaea are not detectable. Carbon flow reconstructions based on stable isotopic compositions of whole archaeal cells, intact archaeal membrane lipids, and other sedimentary carbon pools indicate that these Archaea assimilate sedimentary organic compounds other than methane even though methanotrophy accounts for a major fraction of carbon cycled in these ecosystems. Oxidation of methane by members of Marine Benthic Group B and the Miscellaneous Crenarchaeotal Group without assimilation of methane-carbon provides a plausible explanation. Maintenance energies of these subsurface communities appear to be orders of magnitude lower than minimum values known from laboratory observations, and ecosystem-level carbon budgets suggest that community turnover times are on the order of 100 -2,000 years. Our study provides clues about the metabolic functionality of two cosmopolitan groups of uncultured Archaea.anaerobic methanotrophy ͉ deep biosphere ͉ FISH-secondary ion MS ͉ intact polar lipids ͉ stable carbon isotopes

The subseafloor marine biosphere may be one of the largest reservoirs of microbial biomass on Earth and has recently been the subject of debate in terms of the composition of its microbial inhabitants, particularly on sediments from the Peru Margin. A metagenomic analysis was made by using whole-genome amplification and pyrosequencing of sediments from Ocean Drilling Program Site 1229 on the Peru Margin to further explore the microbial diversity and overall community composition within this environment. A total of 61.9 Mb of genetic material was sequenced from sediments at horizons 1, 16, 32, and 50 m below the seafloor. These depths include sediments from both primarily sulfate-reducing methane-generating regions of the sediment column. Many genes of the annotated genes, including those encoding ribosomal proteins, corresponded to those from the Chloroflexi and Euryarchaeota. However, analysis of the 16S smallsubunit ribosomal genes suggests that Crenarchaeota are the abundant microbial member. Quantitative PCR confirms that uncultivated Crenarchaeota are indeed a major microbial group in these subsurface samples. These findings show that the marine subsurface is a distinct microbial habitat and is different from environments studied by metagenomics, especially because of the predominance of uncultivated archaeal groups.Archaea ͉ Chloroflexi ͉ marine sediment ͉ quantitative PCR

We studied a sample from the GISP 2 (Greenland Ice Sheet Project) ice core to determine the diversity and survival of microorganisms trapped in the ice at least 120,000 years ago. Previously, we examined the phylogenetic relationships among 16S ribosomal DNA (rDNA) sequences in a clone library obtained by PCR amplification from genomic DNA extracted from anaerobic enrichments. Here we report the isolation of nearly 800 aerobic organisms that were grouped by morphology and amplified rDNA restriction analysis patterns to select isolates for further study. The phylogenetic analyses of 56 representative rDNA sequences showed that the isolates belonged to four major phylogenetic groups: the high-G؉C gram-positives, low-G؉C grampositives, Proteobacteria, and the Cytophaga-Flavobacterium-Bacteroides group. The most abundant and diverse isolates were within the high-G؉C gram-positive cluster that had not been represented in the clone library. The Jukes-Cantor evolutionary distance matrix results suggested that at least 7 isolates represent new species within characterized genera and that 49 are different strains of known species. The isolates were further categorized based on the isolation conditions, temperature range for growth, enzyme activity, antibiotic resistance, presence of plasmids, and strain-specific genomic variations. A significant observation with implications for the development of novel and more effective cultivation methods was that preliminary incubation in anaerobic and aerobic liquid prior to plating on agar media greatly increased the recovery of CFU from the ice core sample.

The abundant microbial population in a 3,043-m-deep Greenland glacier ice core was dominated by ultrasmall cells (<0.1 m 3 ) that may represent intrinsically small organisms or starved, minute forms of normalsized microbes. In order to examine their diversity and obtain isolates, we enriched for ultrasmall psychrophiles by filtering melted ice through filters with different pore sizes, inoculating anaerobic low-nutrient liquid media, and performing successive rounds of filtrations and recultivations at 5°C. Melted ice filtrates, cultures, and isolates were analyzed by scanning electron microscopy, flow cytometry, cultivation, and molecular methods. The results confirmed that numerous cells passed through 0.4-m, 0.2-m, and even 0.1-m filters. Interestingly, filtration increased cell culturability from the melted ice, yielding many isolates related to high-G؉C gram-positive bacteria. Comparisons between parallel filtered and nonfiltered cultures showed that (i) the proportion of 0.2-m-filterable cells was higher in the filtered cultures after short incubations but this difference diminished after several months, (ii) more isolates were obtained from filtered (1,290 isolates) than from nonfiltered (447 isolates) cultures, and (iii) the filtration and liquid medium cultivation increased isolate diversity (Proteobacteria; Cytophaga-Flavobacteria-Bacteroides; high-G؉C gram-positive; and spore-forming, low-G؉C gram-positive bacteria). Many isolates maintained their small cell sizes after recultivation and were phylogenetically novel or related to other ultramicrobacteria. Our filtration-cultivation procedure, combined with long incubations, enriched for novel ultrasmall-cell isolates, which is useful for studies of their metabolic properties and mechanisms for long-term survival under extreme conditions.

The examination of microorganisms in glacial ice cores allows the phylogenetic relationships of organisms frozen for thousands of years to be compared with those of current isolates. We developed a method for aseptically sampling a sediment-containing portion of a Greenland ice core that had remained at ؊9°C for over 100,000 years. Epifluorescence microscopy and flow cytometry results showed that the ice sample contained over 6 ؋ 10 7 cells/ml. Anaerobic enrichment cultures inoculated with melted ice were grown and maintained at ؊2°C. Genomic DNA extracted from these enrichments was used for the PCR amplification of 16S rRNA genes with bacterial and archaeal primers and the preparation of clone libraries. Approximately 60 bacterial inserts were screened by restriction endonuclease analysis and grouped into 27 unique restriction fragment length polymorphism types, and 24 representative sequences were compared phylogenetically. Diverse sequences representing major phylogenetic groups including alpha, beta, and gamma Proteobacteria as well as relatives of the Thermus, Bacteroides, Eubacterium, and Clostridium groups were found. Sixteen clone sequences were closely related to those from known organisms, with four possibly representing new species. Seven sequences may reflect new genera and were most closely related to sequences obtained only by PCR amplification. One sequence was over 12% distant from its closest relative and may represent a novel order or family. These results show that phylogenetically diverse microorganisms have remained viable within the Greenland ice core for at least 100,000 years.

SummaryThis study presents comparative geochemical, microbiological and molecular analyses of Greenland GISP2 ice core samples representing different depths, ages, deposition climates, in situ temperatures, and gas and ionic compositions. Our goal was to determine whether specific organisms, preserved at different depths, correlate with past climate characteristics recorded chronologically in ice layers.

No abstract

A psychrophilic gram-positive isolate was obtained from Antarctic Dry Valley soil. It utilized lactose, had a rod-coccus cycle, and contained lysine as the diamino acid in its cell wall. Consistent with these physiological traits, the 16S ribosomal DNA sequence showed that it was phylogenetically related to other Arthrobacter species. A gene (bgaS) encoding a family 2 ␤-galactosidase was cloned from this organism into an Escherichia coli host. Preliminary results showed that the enzyme was cold active (optimal activity at 18°C and 50% activity remaining at 0°C) and heat labile (inactivated within 10 min at 37°C). To enable rapid purification, vectors were constructed adding histidine residues to the BgaS enzyme and its E. coli LacZ counterpart, which was purified for comparison. The His tag additions reduced the specific activities of both ␤-galactosidases but did not alter the other characteristics of the enzymes. Kinetic studies using o-nitrophenyl-␤-D-galactopyranoside showed that BgaS with and without a His tag had greater catalytic activity at and below 20°C than the comparable LacZ ␤-galactosidases. The BgaS heat lability was investigated by ultracentrifugation, where the active enzyme was a homotetramer at 4°C but dissociated into inactive monomers at 25°C. Comparisons of family 2 ␤-galactosidase amino acid compositions and modeling studies with the LacZ structure did not mimic suggested trends for conferring enzyme flexibility at low temperatures, consistent with the changes affecting thermal adaptation being localized and subtle. Mutation studies of the BgaS enzyme should aid our understanding of such specific, localized changes affecting enzyme thermal properties.Glycosyl hydrolases (EC 3.2.1 to 3.2.3) cleave the glycosidic bond between two or more carbohydrates or between a carbohydrate and another moiety. Traditionally, glycosyl hydrolases were classified based on functional similarity. More recently, however, Henrissat and his coworkers have organized these enzymes into 90 glycosyl hydrolase families characterized by hydrophobicity plots, amino acid sequence similarities, and reaction mechanisms (10-12). This system also identifies possible common structural domains, thereby defining evolutionary connections and suggesting reaction mechanisms for the glycosyl hydrolases. Based on these criteria, the once-unified group of enzymes that exhibit ␤-galactosidase activity (EC 3.2.1.23) are now subdivided into four different families: 1, 2, 35, and 42.Of these, the best studied is family 2, which includes the well-characterized ␤-galactosidase from Escherichia coli that is encoded by the lacZ gene. Although there is considerable information about the regulation (1), biochemistry (18,23,35,47), reaction mechanism (17, 45), and structure (16) of this LacZ ␤-galactosidase, few other ␤-galactosidases within this family have been characterized biochemically (4, 7, 13-15, 26, 27, 43), while most examples exist only as a published sequence. Because of the emphasis on the E. coli LacZ enzyme, opportunities to lea...