Project description: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.
Project description:Molecular phylogenetic surveys have recently revealed an ecologically widespread crenarchaeal group that inhabits cold and temperate terrestrial and marine environments. To date these organisms have resisted isolation in pure culture, and so their phenotypic and genotypic characteristics remain largely unknown. To characterize these archaea, and to extend methodological approaches for characterizing uncultivated microorganisms, we initiated genomic analyses of the nonthermophilic crenarchaeote Cenarchaeum symbiosum found living in association with a marine sponge, Axinella mexicana. Complex DNA libraries derived from the host-symbiont population yielded several large clones containing the ribosomal operon from C. symbiosum. Unexpectedly, cloning and sequence analysis revealed the presence of two closely related variants that were consistently found in the majority of host individuals analyzed. Homologous regions from the two variants were sequenced and compared in detail. The variants exhibit >99.2% sequence identity in both small- and large-subunit rRNA genes and they contain homologous protein-encoding genes in identical order and orientation over a 28-kbp overlapping region. Our study not only indicates the potential for characterizing uncultivated prokaryotes by genome sequencing but also identifies the primary complication inherent in the approach: the widespread genomic microheterogeneity in naturally occurring prokaryotic populations.
Project description:Marine sponges represent one of the few eukaryotic groups that ubiquitously harbor symbiotic members of the Thaumarchaeota, which are important chemoautotrophic ammonia-oxidizers in many environments. However in most studies, direct demonstration of ammonia-oxidation by these archaea within sponges is lacking, and little is known about sponge-specific adaptations of archaeal ammonia oxidizers (AOA). In this study, we characterized the thaumarchaeal symbiont of the marine sponge Ianthella basta using metaproteogenomics, fluorescence in situ hybridization, qPCR and direct isotope-based functional assays. We demonstrate that the I. basta symbiont is not closely related to other genomically sequenced sponge AOA and is a member of a new genus. “Candidatus Nitrosospongia bastadiensis” is an abundant symbiont that is solely responsible for nitrite formation from ammonia in I. basta that surprisingly does not harbor nitrite-oxidizing microbes. Consistently, Ca N. bastadiensis encodes and expresses the genetic repertoire required for chemolithoautotrophic ammonia oxidation. Furthermore, we show that this AOA is equipped with an expanded set of extracellular subtilisin-like proteases, a metalloprotease unique among archaea, as well as a putative branched-chain amino acid ABC transporter. This repertoire is strongly indicative of a mixotrophic lifestyle and is (with slight variations) also found in other sponge-associated, but not in free-living AOA. We predict that this feature as well as an expanded and unique set of secreted serpins (protease inhibitors), a unique array of eukaryotic-like proteins, and a DNA-phosporothioation system likely involved in defense against foreign DNA, represent important adaptations of AOA to life within these ancient filter-feeding animals.
Project description:Cenarchaeum symbiosum, an archaeon which lives in specific association with a marine sponge, belongs to a recently recognized nonthermophilic crenarchaeotal group that inhabits diverse cold and temperate environments. Nonthermophilic crenarchaeotes have not yet been obtained in laboratory culture, and so their phenotypic characteristics have been inferred solely from their ecological distribution. Here we report on the first protein to be characterized from one of these organisms. The DNA polymerase gene of C. symbiosum was identified in the vicinity of the rRNA operon on a large genomic contig. Its deduced amino acid sequence is highly similar to those of the archaeal family B (alpha-type) DNA polymerases. It shared highest overall sequence similarity with the crenarchaeal DNA polymerases from the extreme thermophiles Sulfolobus acidocaldarius and Pyrodictium occultum (54% and 53%, respectively). The conserved motifs of B (alpha-)-type DNA polymerases and 3'-5' exonuclease were identified in the 845-amino-acid sequence. The 96-kDa protein was expressed in Escherichia coli and purified with affinity tags. It exhibited its highest specific activity with gapped-duplex (activated) DNA as the substrate. Single-strand- and double-strand-dependent 3'-5' exonuclease activity was detected, as was a marginal 5'-3' exonuclease activity. The enzyme was rapidly inactivated at temperatures higher than 40 degrees C, with a half-life of 10 min at 46 degrees C. It was found to be less thermostable than polymerase I of E. coli and is substantially more heat labile than its most closely related homologs from thermophilic and hyperthermophilic crenarchaeotes. Although phylogenetic studies suggest a thermophilic ancestry for C. symbiosum and its relatives, our biochemical analysis of the DNA polymerase is consistent with the postulated nonthermophilic phenotype of these crenarchaeotes, to date inferred solely from their ecological distribution.
Project description:Archaea, one of the three major domains of extant life, was thought to comprise predominantly microorganisms that inhabit extreme environments, inhospitable to most Eucarya and Bacteria. However, molecular phylogenetic surveys of native microbial assemblages are beginning to indicate that the evolutionary and physiological diversity of Archaea is far greater than previously supposed. We report here the discovery and preliminary characterization of a marine archaeon that inhabits the tissues of a temperate water sponge. The association was specific, with a single crenarchaeal phylotype inhabiting a single sponge host species. To our knowledge, this partnership represents the first described symbiosis involving Crenarchaeota. The symbiotic archaeon grows well at temperatures of 10 degrees C, over 60 degrees C below the growth temperature optimum of any cultivated species of Crenarchaeota. Archaea have been generally characterized as microorganisms that inhabit relatively circumscribed niches, largely high-temperature anaerobic environments. In contrast, data from molecular phylogenetic surveys, including this report, suggest that some crenarchaeotes have diversified considerably and are found in a wide variety of lifestyles and habitats. We present here the identification and initial description of Cenarchaeum symbiosum gen. nov., sp. nov., a symbiotic archaeon closely related to other nonthermophilic crenarchaeotes that inhabit diverse marine and terrestrial environments.