A first insight into the occurrence and expression of functional amoA and accA genes of autotrophic and ammonia-oxidizing bathypelagic Crenarchaeota of Tyrrhenian Sea

The autotrophic and ammonia-oxidizing crenarchaeal assemblage at offshore site located in the deep Mediterranean (Tyrrhenian Sea, depth 3000 m) water was studied by PCR amplification of the key functional genes involved in energy (ammonia mono-oxygenase alpha subunit, amoA) and central metabolism (acetyl-CoA carboxylase alpha subunit, accA). Using two recently annotated genomes of marine crenarchaeons, an initial set of primers targeting archaeal accA-like genes was designed. Approximately 300 clones were analyzed, of which 100% of amoA library and almost 70% of accA library were unambiguously related to the corresponding genes from marine Crenarchaeota. Even though the acetyl-CoA carboxylase is phylogenetically not well conserved and the remaining clones were affiliated to various bacterial acetyl-CoA/propionyl-CoA carboxylase genes, the pool of archaeal sequences was applied for development of quantitative PCR analysis of accA-like distribution using TaqMan® methodolgy. The archaeal accA gene fragments, together with alignable gene fragments from the Sargasso Sea and North Pacific Subtropical Gyre (ALOHA Station) metagenome databases, were analyzed by multiple sequence alignment. Two accA-like sequences, found in ALOHA Station at the depth of 4000 m, formed a deeply branched clade with 64% of all archaeal Tyrrhenian clones. No close relatives for residual 36% of clones, except of those recovered from Eastern Mediterranean, was found, suggesting the existence of a specific lineage of the crenarchaeal accA genes in deep Mediterranean water. Alignment of Mediterranean amoA sequences defined four cosmopolitan phylotypes of Crenarchaeota putative ammonia mono-oxygenase subunit A gene occurring in the water sample from the 3000 m depth. Without exception all phylotypes fell into Deep Marine Group I cluster that contain the vast majority of known sequences recovered from global deep-sea environment. Remarkably, three phylotypes accounted for 91% of all Mediterranean amoA clones and corresponded to the sequences retrieved from the less deep compartments of the world's ocean, most likely reflecting the higher temperature at the depth of the Mediterranean Sea. In order to verify whether these phylotypes might represent important Crenarchaeota in the functioning of the Mediterranean bathypelagic ecosystem, expression of crenarchaeal amoA gene was monitored by direct RNA retrieval and following analysis of amoA-related mRNA transcripts. Surprisingly, all mRNA-derived sequences formed a tight monophyletic group, which fell into large Shallow Marine Group I cluster with sequences retrieved from shallow (up to 200 m) waters, sediments and corals. This group was not detected in DNA-based clone library, obviously, due to an overwhelming dominance of the Deep Marine Group I. The failure to recover the amoA transcripts, related to Deep Marine Group I of Crenarchaeota, was unanticipated and likely resulted from the physiology of these strongly adapted deep-sea organisms. As far as all seawater samples were treated on-board under atmospheric pressure conditions and sunlight, the decompression and/or photoinhibition likely affected their metabolic activity, followed by the strong decay of gene expression.