José R. de la Torre

Isolation of an autotrophic ammonia-oxidizing marine archaeon

For years, microbiologists characterized the Archaea as obligate extremophiles that thrive in environments too harsh for other organisms. The limited physiological diversity among cultivated Archaea suggested that these organisms were metabolically constrained to a few environmental niches. For instance, all Crenarchaeota that are currently cultivated are sulphur-metabolizing thermophiles. However, landmark studies using cultivation-independent methods uncovered vast numbers of Crenarchaeota in cold oxic ocean waters. Subsequent molecular surveys demonstrated the ubiquity of these low-temperature Crenarchaeota in aquatic and terrestrial environments. The numerical dominance of marine Crenarchaeota—estimated at 1028 cells in the worlds oceans—suggests that they have a major role in global biogeochemical cycles. Indeed, isotopic analyses of marine crenarchaeal lipids suggest that these planktonic Archaea fix inorganic carbon. Here we report the isolation of a marine crenarchaeote that grows chemolithoautotrophically by aerobically oxidizing ammonia to nitrite—the first observation of nitrification in the Archaea. The autotrophic metabolism of this isolate, and its close phylogenetic relationship to environmental marine crenarchaeal sequences, suggests that nitrifying marine Crenarchaeota may be important to global carbon and nitrogen cycles.

Netrins are diffusible chemotropic factors for commissural axons in the embryonic spinal cord.

The guidance of axons to their targets in the developing nervous system is believed to involve diffusible chemotropic factors secreted by target cells. Floor plate cells at the ventral midline of the spinal cord secrete a diffusible factor or factors that promotes the outgrowth of spinal commissural axons and attracts these axons in vitro. Two membrane-associated proteins isolated from brain, netrin-1 and netrin-2, possess commissural axon outgrowth-promoting activity. We show here that netrin-1 RNA is expressed by floor plate cells, whereas netrin-2 RNA is detected at lower levels in the ventral two-thirds of the spinal cord, but not the floor plate. Heterologous cells expressing recombinant netrin-1 or netrin-2 secrete diffusible forms of the proteins and can attract commissural axons at a distance. These results show that netrin-1 is a chemotropic factor expressed by floor plate cells and suggest that the two netrin proteins guide commissural axons in the developing spinal cord.

Ammonia oxidation kinetics determine niche separation of nitrifying Archaea and Bacteria

The discovery of ammonia oxidation by mesophilic and thermophilic Crenarchaeota and the widespread distribution of these organisms in marine and terrestrial environments indicated an important role for them in the global nitrogen cycle. However, very little is known about their physiology or their contribution to nitrification. Here we report oligotrophic ammonia oxidation kinetics and cellular characteristics of the mesophilic crenarchaeon ‘Candidatus Nitrosopumilus maritimus’ strain SCM1. Unlike characterized ammonia-oxidizing bacteria, SCM1 is adapted to life under extreme nutrient limitation, sustaining high specific oxidation rates at ammonium concentrations found in open oceans. Its half-saturation constant (Km = 133 nM total ammonium) and substrate threshold (≤10 nM) closely resemble kinetics of in situ nitrification in marine systems and directly link ammonia-oxidizing Archaea to oligotrophic nitrification. The remarkably high specific affinity for reduced nitrogen (68,700 l per g cells per h) of SCM1 suggests that Nitrosopumilus-like ammonia-oxidizing Archaea could successfully compete with heterotrophic bacterioplankton and phytoplankton. Together these findings support the hypothesis that nitrification is more prevalent in the marine nitrogen cycle than accounted for in current biogeochemical models.

Genomic analysis of the uncultivated marine crenarchaeote Cenarchaeum symbiosum

Crenarchaeota are ubiquitous and abundant microbial constituents of soils, sediments, lakes, and ocean waters. To further describe the cosmopolitan nonthermophilic Crenarchaeota, we analyzed the genome sequence of one representative, the uncultivated sponge symbiont Cenarchaeum symbiosum. C. symbiosum genotypes coinhabiting the same host partitioned into two dominant populations, corresponding to previously described a- and b-type ribosomal RNA variants. Although they were syntenic, overlapping a- and b-type ribotype genomes harbored significant variability. A single tiling path comprising the dominant a-type genotype was assembled and used to explore the genomic properties of C. symbiosum and its planktonic relatives. Of 2,066 ORFs, 55.6% matched genes with predicted function from previously sequenced genomes. The remaining genes partitioned between functional RNAs (2.4%) and hypotheticals (42%) with limited homology to known functional genes. The latter category included some genes likely involved in the archaeal–sponge symbiotic association. Conversely, 525 C. symbiosum ORFs were most highly similar to sequences from marine environmental genomic surveys, and they apparently represent orthologous genes from free-living planktonic Crenarchaeota. In total, the C. symbiosum genome was remarkably distinct from those of other known Archaea and shared many core metabolic features in common with its free-living planktonic relatives.

Turning of Retinal Growth Cones in a Netrin-1 Gradient Mediated by the Netrin Receptor DCC

Netrin-1 promotes outgrowth of axons in vitro through the receptor Deleted in Colorectal Cancer (DCC) and elicits turning of axons within embryonic explants when presented as a point source. It is not known whether netrin-1 alone can elicit turning nor whether DCC mediates the turning response. We show that Xenopus retinal ganglion cell growth cones orient rapidly toward a pipette ejecting netrin-1, an effect blocked by antibodies to DCC. In vitro, netrin-1 induces a complex growth cone morphology reminiscent of that at the optic nerve head, a site of netrin-1 expression in vivo. These results demonstrate that netrin-1 can function alone to induce turning, implicate DCC in this response, and support the idea that netrin-1 contributes to steering axons out of the retina.

Physiology and Diversity of Ammonia-Oxidizing Archaea

The discovery of ammonia-oxidizing archaea (AOA), now generally recognized to exert primary control over ammonia oxidation in terrestrial, marine, and geothermal habitats, necessitates a reassessment of the nitrogen cycle. In particular, the unusually high affinity of marine and terrestrial AOA for ammonia indicates that this group may determine the oxidation state of nitrogen available to associated micro- and macrobiota, altering our current understanding of trophic interactions. Initial comparative genomics and physiological studies have revealed a novel, and as yet unresolved, primarily copper-based pathway for ammonia oxidation and respiration distinct from that of known ammonia-oxidizing bacteria and possibly relevant to the production of atmospherically active nitrogen oxides. Comparative studies also provide compelling evidence that the lineage of Archaea with which the AOA affiliate is sufficiently divergent to justify the creation of a novel phylum, the Thaumarchaeota.

Microbial diversity of cryptoendolithic communities from the McMurdo Dry Valleys, Antarctica.

ABSTRACT In the McMurdo Dry Valleys of Antarctica, microorganisms colonize the pore spaces of exposed rocks and are thereby protected from the desiccating environmental conditions on the surface. These cryptoendolithic communities have received attention in microscopy and culture-based studies but have not been examined by molecular approaches. We surveyed the microbial biodiversity of selected cryptoendolithic communities by analyzing clone libraries of rRNA genes amplified from environmental DNA. Over 1,100 individual clones from two types of cryptoendolithic communities, cyanobacterium dominated and lichen dominated, were analyzed. Clones fell into 51 relatedness groups (phylotypes) with ≥98% rRNA sequence identity (46 bacterial and 5 eucaryal). No representatives of Archaea were detected. No phylotypes were shared between the two classes of endolithic communities studied. Clone libraries based on both types of communities were dominated by a relatively small number of phylotypes that, because of their relative abundance, presumably represent the main primary producers in these communities. In the lichen-dominated community, three rRNA sequences, from a fungus, a green alga, and a chloroplast, of the types known to be associated with lichens, accounted for over 70% of the clones. This high abundance confirms the dominance of lichens in this community. In contrast, analysis of the supposedly cyanobacterium-dominated community indicated, in addition to cyanobacteria, at least two unsuspected organisms that, because of their abundance, may play important roles in the community. These included a member of the α subdivision of the Proteobacteria that potentially is capable of aerobic anoxygenic photosynthesis and a distant relative of Deinococcus that defines, along with other Deinococcus-related sequences from Antarctica, a new clade within the Thermus-Deinococcus bacterial phylogenetic division.

Proteorhodopsin genes are distributed among divergent marine bacterial taxa

Proteorhodopsin (PR) is a retinal-binding bacterial integral membrane protein that functions as a light-driven proton pump. The gene encoding this photoprotein was originally discovered on a large genome fragment derived from an uncultured marine γ-proteobacterium of the SAR86 group. Subsequently, many variants of the PR gene have been detected in marine plankton, via PCR-based gene surveys. It has not been clear, however, whether these different PR genes are widely distributed among different bacterial groups, or whether they have a restricted taxonomic distribution. We report here comparative analyses of PR-bearing genomic fragments recovered directly from planktonic bacteria inhabiting the California coast, the central Pacific Ocean, and waters offshore the Antarctica Peninsula. Sequence analysis of an Antarctic genome fragment harboring PR (ANT32C12) revealed moderate conservation in gene order and identity, compared with a previously reported PR-containing genome fragment from a Monterey Bay γ-proteobacterium (EBAC31A08). Outside the limited region of synteny shared between these clones, however, no significant DNA or protein identity was evident. Analysis of a third PR-containing genome fragment (HOT2C01) from the North Pacific subtropical gyre showed even more divergence from the γ-proteobacterial PR-flanking region. Subsequent phylogenetic and comparative genomic analyses revealed that the Central North Pacific PR-containing genome fragment (HOT2C01) originated from a planktonic α-proteobacterium. These data indicate that PR genes are distributed among a variety of divergent marine bacterial taxa, including both α- and γ-proteobacteria. Our analyses also demonstrate the utility of cultivation-independent comparative genomic approaches for assessing gene content and distribution in naturally occurring microbes.

Abundance of Ammonia-Oxidizing Archaea and Bacteria along an Estuarine Salinity Gradient in Relation to Potential Nitrification Rates

ABSTRACT Abundance of ammonia-oxidizing Archaea (AOA) was found to be always greater than that of ammonia-oxidizing Bacteria along an estuarine salinity gradient, and AOA abundance was highest at intermediate salinity. However, AOA abundance did not correlate with potential nitrification rates. This lack of correlation may be due to methodological limitations or alternative energy sources.

Cdv-based cell division and cell cycle organization in the thaumarchaeon Nitrosopumilus maritimus

Cell division is mediated by different mechanisms in different evolutionary lineages. While bacteria and euryarchaea utilize an FtsZ‐based mechanism, most crenarchaea divide using the Cdv system, related to the eukaryotic ESCRT‐III machinery. Intriguingly, thaumarchaeal genomes encode both FtsZ and Cdv protein homologues, raising the question of their division mode. Here, we provide evidence indicating that Cdv is the primary division system in the thaumarchaeon Nitrosopumilus maritimus. We also show that the cell cycle is differently organized as compared to hyperthermophilic crenarchaea, with a longer pre‐replication phase and a shorter post‐replication stage. In particular, the time required for chromosome replication is remarkably extensive, 15–18 h, indicating a low replication rate. Further, replication did not continue to termination in a significant fraction of N. maritimus cell populations following substrate depletion. Both the low replication speed and the propensity for replication arrest are likely to represent adaptations to extremely oligotrophic environments. The results demonstrate that thaumarchaea, crenarchaea and euryarchaea display differences not only regarding phylogenetic affiliations and gene content, but also in fundamental cellular and physiological characteristics. The findings also have implications for evolutionary issues concerning the last archaeal common ancestor and the relationship between archaea and eukaryotes.